43 results on '"Deng, H."'
Search Results
2. A resonance Raman study of the C=N configurations of octopus rhodopsin, bathorhodopsin, and isorhodopsin
- Author
-
Huang, L., Deng, H., Weng, G., Koutalos, Y., Ebrey, T., Groesbeek, M., Lugtenburg, J., Tsuda, M., and Callender, R.H.
- Subjects
Raman spectroscopy -- Usage ,Rhodopsin -- Analysis ,Biological sciences ,Chemistry - Abstract
Raman spectra studies reveal that the C=N configuration is anti in octopus rhodopsin and bathorhodopsin while more work is necessary for determining the C=N bond configuration in isorhodopsin. Octopus bathorhodopsin is 11-trans and anti about the Schiff base. The conformation of rhodopsin is 11-cis, 12-s-trans and the Schiff base C=N bond is anti. The spectra of octopus rhodopsin are different from those of bovine rhodopsin due to changes in the C14-C15 and C12-C13 stretch motions and suggest large protein perturbations on the C11=C12-C13 moiety due to electrostatic or steric interactions.
- Published
- 1996
3. Raman Difference Studies of GDP and GTP Binding to c-Harvey ras
- Author
-
Wang, J. H., primary, Xiao, D. G., additional, Deng, H., additional, Webb, Martin R., additional, and Callender, Robert, additional
- Published
- 1998
- Full Text
- View/download PDF
4. Resonance Raman spectroscopy of an ultraviolet sensitive insect rhodopsin
- Author
-
Pande, C., primary, Deng, H., additional, Rath, P., additional, Callender, R. H., additional, and Schwemer, J., additional
- Published
- 1987
- Full Text
- View/download PDF
5. A study of the Schiff base mode in bovine rhodopsin and bathorhodopsin
- Author
-
Deng, H., primary and Callender, R. H., additional
- Published
- 1987
- Full Text
- View/download PDF
6. Structural Characterization of the Interaction between the α M I-Domain of the Integrin Mac-1 (α M β 2 ) and the Cytokine Pleiotrophin.
- Author
-
Feng W, Nguyen H, Shen D, Deng H, Jiang Z, Podolnikova N, Ugarova T, and Wang X
- Subjects
- Carrier Proteins metabolism, Cytokines metabolism, Humans, Hydrophobic and Hydrophilic Interactions, Macrophage-1 Antigen metabolism, Nuclear Magnetic Resonance, Biomolecular, Protein Domains, Static Electricity, Carrier Proteins chemistry, Cytokines chemistry, Macrophage-1 Antigen chemistry, Models, Molecular
- Abstract
Integrin Mac-1 (α
M β2 ) is an adhesion receptor vital to many functions of myeloid leukocytes. It is also the most promiscuous member of the integrin family capable of recognizing a broad range of ligands. In particular, its ligand-binding αM I-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αM I-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of the αM I-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through NMR chemical shift perturbation analysis, cross saturation, NOESY, and mutagenesis studies, we found the interaction between the αM I-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5-β5 loop of αM I-domain. The observation that increased ionic strength weakens the interaction between the proteins indicates electrostatic forces may also play a significant role in the binding. On the basis of the results from these experiments, we formulated a model of the interaction between the αM I-domain and PTN.- Published
- 2021
- Full Text
- View/download PDF
7. Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase from Bacillus stearothermophilus.
- Author
-
Nie B, Lodewyks K, Deng H, Desamero RZ, and Callender R
- Subjects
- Animals, Binding Sites, Catalytic Domain, Kinetics, Protein Binding, Protein Conformation, Substrate Specificity, Swine, Thermodynamics, Geobacillus stearothermophilus enzymology, L-Lactate Dehydrogenase chemistry, L-Lactate Dehydrogenase metabolism, NAD metabolism
- Abstract
Laser-induced temperature-jump relaxation spectroscopy was used to study the active site mobile-loop dynamics found in the binding of the NADH nucleotide cofactor and oxamate substrate mimic to lactate dehydrogenase in Bacillus stearothermophilus thermophilic bacteria (bsLDH). The kinetic data can be best described by a model in which NADH can bind only to the open-loop apoenzyme, oxamate can bind only to the bsLDH·NADH binary complex in the open-loop conformation, and oxamate binding is followed by closing of the active site loop preventing oxamate unbinding. The open and closed states of the loop are in dynamic equilibrium and interconvert on the submillisecond time scale. This interconversion strongly accelerates with an increase in temperature because of significant enthalpy barriers. Binding of NADH to bsLDH results in minor changes of the loop dynamics and does not shift the open-closed equilibrium, but binding of the oxamate substrate mimic shifts this equilibrium to the closed state. At high excess oxamate concentrations where all active sites are nearly saturated with the substrate mimic, all active site mobile loops are mainly closed. The observed active-loop dynamics for bsLDH is very similar to that previously observed for pig heart LDH., Competing Interests: The authors declare no competing financial interest.
- Published
- 2016
- Full Text
- View/download PDF
8. Energy landscape of the Michaelis complex of lactate dehydrogenase: relationship to catalytic mechanism.
- Author
-
Peng HL, Deng H, Dyer RB, and Callender R
- Subjects
- Animals, Catalysis, Catalytic Domain, NAD chemistry, Pyruvic Acid chemistry, Spectroscopy, Fourier Transform Infrared methods, Stochastic Processes, Swine, Energy Metabolism physiology, Hydro-Lyases chemistry, Hydro-Lyases metabolism
- Abstract
Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the "live" reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD(+)·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate's C2═O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2═O group and active site residues, as shown by the observation of at least four C2═O stretch bands indicating varying degrees of C2═O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution.
- Published
- 2014
- Full Text
- View/download PDF
9. Large scale dynamics of the Michaelis complex in Bacillus stearothermophilus lactate dehydrogenase revealed by a single-tryptophan mutant study.
- Author
-
Nie B, Deng H, Desamero R, and Callender R
- Subjects
- Catalytic Domain, Geobacillus stearothermophilus genetics, Geobacillus stearothermophilus metabolism, Kinetics, L-Lactate Dehydrogenase chemistry, Models, Molecular, NAD chemistry, NAD metabolism, Oxamic Acid chemistry, Oxamic Acid metabolism, Point Mutation, Protein Conformation, Temperature, Tryptophan chemistry, Tryptophan metabolism, Geobacillus stearothermophilus enzymology, L-Lactate Dehydrogenase genetics, L-Lactate Dehydrogenase metabolism, Tryptophan genetics
- Abstract
Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophilic lactate dehydrogenase, bsLDH·NADH·oxamate, were studied with site specific resolution by laser-induced temperature jump relaxation spectroscopy with a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the millisecond to microsecond time scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called "loop motion" is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on the catalytic mechanism of bsLDH is discussed.
- Published
- 2013
- Full Text
- View/download PDF
10. Pyrophosphate activation in hypoxanthine--guanine phosphoribosyltransferase with transition state analogue.
- Author
-
Deng H, Callender R, Schramm VL, and Grubmeyer C
- Subjects
- Binding Sites, Catalytic Domain, Enzyme Activation, Hydrogen Bonding, Hypoxanthine chemistry, Models, Molecular, Substrate Specificity, Diphosphates pharmacology, Hypoxanthine Phosphoribosyltransferase chemistry, Hypoxanthine Phosphoribosyltransferase pharmacology
- Abstract
Isotope-edited difference Raman and FTIR studies complemented by ab initio calculations have been applied to the transition state analogue complex of HGPRT.ImmHP.MgPP(i) to determine the ionic states of the 5'-phosphate moiety of ImmHP and of PP(i). These measurements characterize electrostatic interactions within the enzyme active site as deduced from frequency shifts of the phosphate groups. The bound 5'-phosphate moiety of ImmHP is dianionic, and this phosphate group exists in two different conformations within the protein complex. In one conformation, a hydrogen bond between the 5'-phosphate of ImmHP and the OH group of Tyr104 in the catalytic loop appears to be stronger. With the stronger H-bond, the OH of Tyr104 approaches one of the P..O bonds from the bridging oxygen side to cause distortion of the PO(3) moiety, as indicated by a lowered symmetric P..O stretch frequency. The asymmetric stretch frequencies are similar in both phosphate conformations. Bound PP(i) in this complex is fully ionized to P(2)O(7)(4-). Bond frequency changes for bound PP(i) indicate coordination to Mg(2+) ions but show no indication of significant P..O bond polarization. Extrapolation of these results to reaction coordinate motion for HGPRT suggests that bond formation between C1' of the nucleotide ribose and the oxygen of PP(i) is accomplished by migration of the ribocation toward immobilized pyrophosphate.
- Published
- 2010
- Full Text
- View/download PDF
11. Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues.
- Author
-
Rinaldo-Matthis A, Wing C, Ghanem M, Deng H, Wu P, Gupta A, Tyler PC, Evans GB, Furneaux RH, Almo SC, Wang CC, and Schramm VL
- Subjects
- Adenine chemistry, Adenine pharmacology, Adenosine analogs & derivatives, Animals, Catalytic Domain, Crystallization, Crystallography, X-Ray, Models, Molecular, Substrate Specificity, Trichomonas vaginalis enzymology, Adenine analogs & derivatives, Purine-Nucleoside Phosphorylase antagonists & inhibitors, Purine-Nucleoside Phosphorylase chemistry, Pyrrolidines chemistry, Pyrrolidines pharmacology
- Abstract
Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.
- Published
- 2007
- Full Text
- View/download PDF
12. Engineering of a protein with cyclooxygenase and prostacyclin synthase activities that converts arachidonic acid to prostacyclin.
- Author
-
Ruan KH, Deng H, and So SP
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Catalysis, Cell Line, Cytochrome P-450 Enzyme System chemistry, DNA Primers, Humans, Intramolecular Oxidoreductases chemistry, Prostaglandin-Endoperoxide Synthases chemistry, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Substrate Specificity, Arachidonic Acid metabolism, Cytochrome P-450 Enzyme System metabolism, Epoprostenol metabolism, Intramolecular Oxidoreductases metabolism, Prostaglandin-Endoperoxide Synthases metabolism, Protein Engineering
- Abstract
Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.
- Published
- 2006
- Full Text
- View/download PDF
13. Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations.
- Author
-
Deng H, Cahill SM, Abad JL, Lewandowicz A, Callender RH, Schramm VL, and Jones RA
- Subjects
- Animals, Binding Sites, Catalytic Domain, Cattle, Computational Biology, Humans, Hydrogen Bonding, Hypoxanthine metabolism, Nitrogen Isotopes chemistry, Nuclear Magnetic Resonance, Biomolecular, Purine-Nucleoside Phosphorylase metabolism, Solutions chemistry, Hypoxanthine chemistry, Purine-Nucleoside Phosphorylase chemistry
- Abstract
Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.
- Published
- 2004
- Full Text
- View/download PDF
14. Assignment of downfield proton resonances in purine nucleoside phosphorylase immucillin-H complex by saturation-transferred NOEs.
- Author
-
Deng H, Lewandowicz A, Cahill SM, Furneaux RH, Tyler PC, Girvin ME, Callender RH, and Schramm VL
- Subjects
- Binding, Competitive, Catalysis, Enzyme Activation, Enzyme Inhibitors chemistry, Humans, Hydrogen Bonding, Nuclear Magnetic Resonance, Biomolecular methods, Protein Binding, Purine Nucleosides, Purine-Nucleoside Phosphorylase antagonists & inhibitors, Recombinant Proteins chemistry, Solutions, Thermodynamics, Protons, Purine-Nucleoside Phosphorylase chemistry, Pyrimidinones chemistry, Pyrroles chemistry
- Abstract
Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and alpha-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1', ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4' is protonated to the cation. It binds tightly to the PNPs with a K(d) value 56 pM for human PNP. Previous NMR studies of PNP. ImmH.PO(4) have shown that the N-4' of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (>11 ppm) are specific to the PNP. ImmH.PO(4) complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using (15)N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only approximately 1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP. ImmH.PO(4) complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP. ImmH complex. The approximately 4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to O-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.
- Published
- 2004
- Full Text
- View/download PDF
15. Identification of the residues in the helix F/G loop important to catalytic function of membrane-bound prostacyclin synthase.
- Author
-
Deng H, Wu J, So SP, and Ruan KH
- Subjects
- 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid, Amino Acid Sequence, Animals, Base Sequence, COS Cells, Catalytic Domain genetics, Cytochrome P-450 Enzyme System genetics, DNA genetics, Endoplasmic Reticulum enzymology, Humans, In Vitro Techniques, Intracellular Membranes enzymology, Intramolecular Oxidoreductases genetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Solutions, Substrate Specificity, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System metabolism, Intramolecular Oxidoreductases chemistry, Intramolecular Oxidoreductases metabolism
- Abstract
A topological model of prostaglandin I(2) synthase (PGIS) was created by homology modeling. This model, along with site-specific antibodies and other topology studies, has suggested that the residue(s) within helix F/G loop of PGIS may be involved in forming the substrate access channel and located in a position that influences the membrane-bound PGIS catalytic function (1). To test this hypothesis, we have explored an approach to identify the residues of the helix F/G loop important to enzyme activity of the membrane-bound PGIS by a combination of 2-D NMR experiment and mutagenesis methods. Using the distance measured from the model as a guide, the helix F/G loop was mimicked in a synthetic peptide by introducing a spacer to maintain a distance of about 7 A between the N- and the C-termini (PGIS residues 208 and 230). The peptide was used to interact with the enzyme substrate analogue, U46619. High-resolution 2-D NMR experiments were performed to determine the contacts between the peptide and U46619. The interaction between the constrained F/G loop peptide and U46619 was confirmed by the observation of the conformational changes of the peptide and U46619 using the comparison of the cross-peaks between the NOESY spectra of U46619 with the peptide, without the peptide, and the peptide alone. Through the combination of the 2-D NMR experiments, completed (1)H NMR assignments of the F/G loop segment in the presence and absence of U46619 were obtained, and these data were used to predict the contact residues (Leu214 and Pro215) of the F/G loop with PGIS substrate. The predicted influence of residues on enzyme catalytic activity in membrane-bound environments was confirmed by the mutagenesis of the F/G loop residues of human PGIS. These observations support that the F/G loop is involved in forming the substrate access channel for membrane-bound PGIS and suggests that the NMR experiment-based mutagenesis approach may be applied to study structure and function relationships for other proteins.
- Published
- 2003
- Full Text
- View/download PDF
16. Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy.
- Author
-
Deng H, Callender R, Zhu J, Nguyen KT, and Pei D
- Subjects
- Absorption, Alanine genetics, Amides chemistry, Amino Acid Substitution genetics, Aminopeptidases genetics, Aspartic Acid genetics, Buffers, Catalysis, Chlorides, Cobalt chemistry, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Glutamic Acid genetics, Hydrogen-Ion Concentration, Metalloproteins genetics, Mutagenesis, Site-Directed, Spectroscopy, Fourier Transform Infrared methods, Amidohydrolases, Aminopeptidases chemistry, Glutamic Acid chemistry, Metalloproteins chemistry, Protons
- Abstract
Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria and the organelles of certain eukaryotes. PDF is a novel class of amide hydrolase, which utilizes an Fe2+ ion to effect the hydrolysis of an amide bond. The ferrous ion is tetrahedrally coordinated by two histidines from a conserved HEXXH motif, a cysteine, and a water molecule. In this work, the function of the conserved glutamate (Glu-133 in Escherichia coli PDF) is evaluated by difference FTIR spectroscopic analysis of a Co(II)-substituted E. coli wild-type and E133D mutant PDF. At pH <6, the wild-type enzyme exhibited a relatively sharp C=O stretch band at 1742 cm(-1), which is assigned to the COOH group of Glu-133. The pH titration study and curve fitting to the data revealed a pK(a) of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only approximately 10-fold less active than the wild-type enzyme, a similar pH titration study of the Asp-133 C=O stretch band at 1740 cm(-1) revealed a pK(a) of 10.1. This unusually high pK(a) for a carboxyl group is likely due to its hydrophobic environment and electrostatic repulsion from the metal-bound hydroxide. These results argue that in the active form of E133D PDF, Asp-133 is protonated and therefore acts as a general acid during the decomposition of the tetrahedral intermediate by donating a proton to the leaving amide ion perhaps through a water molecule in the cavity created by the E133D mutation. In contrast, Glu-133 is deprotonated in the active form of wild-type PDF. We propose that Glu-133 acts as a proton shuttle accepting a proton from the metal-bound water and subsequently acts as a general acid during the decomposition of the tetrahedral intermediate.
- Published
- 2002
- Full Text
- View/download PDF
17. Is the PTPase-vanadate complex a true transition state analogue?
- Author
-
Deng H, Callender R, Huang Z, and Zhang ZY
- Subjects
- Binding Sites, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacology, Hydrolysis, Kinetics, Protein Binding, Protein Tyrosine Phosphatases antagonists & inhibitors, Spectrum Analysis, Raman, Vanadates pharmacology, Protein Tyrosine Phosphatases chemistry, Protein Tyrosine Phosphatases metabolism, Vanadates metabolism, Yersinia enzymology
- Abstract
Vanadate can often bind to phosphoryl transfer enzymes to form a trigonal-bipyramidal structure at the active site. The enzyme-vanadate dissociation constants in these enzymes are much lower than those for phosphate. Therefore, enzyme-bound vanadate moieties are often considered as transition state analogues. To test whether the enzyme-vanadate complex is a true transition state analogue beyond the simple geometry and binding affinity arguments and whether the bond orders of the VO bonds in the complex approach those of the PO bonds in the transition state, the binding properties of vanadate in the Yersinia protein-tyrosine phosphatase (PTPase) and its T410A, D356N, W354A, R409K, and D356A mutants have been studied by steady-state kinetic measurements and by difference Raman measurements. The results of the kinetic measurements show no correlation between K(I) and kcat or kcat/K(m) in these mutants. In addition, our analysis of the Raman data shows that the bond order change of the nonbridging V--O bonds in the vanadate complexes does not correlate with the kinetic parameters in a number of PTPase variants as predicted by the transition state binding paradigm. Furthermore, the ionization state of the bound vanadate moiety is not invariant across the PTPase variants studied, and the average bond order of the nonbridging V--O bonds decreased by 0.06-0.07 valence unit in the wild type and all of the mutant PTPases, either in dianionic or in monoanionic form. Thus the complex would resemble an associative transition state, contrary to the previously determined dissociative structure of the transition state. Therefore, it is concluded that vanadate is not a true transition state analogue for the PTPase reactions.
- Published
- 2002
- Full Text
- View/download PDF
18. Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase.
- Author
-
Gulotta M, Deng H, Deng H, Dyer RB, and Callender RH
- Subjects
- Catalysis, Kinetics, L-Lactate Dehydrogenase chemistry, Spectrometry, Fluorescence, Spectrophotometry, Infrared, Spectrophotometry, Ultraviolet, Thermodynamics, L-Lactate Dehydrogenase metabolism
- Abstract
The motions of key residues at the substrate binding site of lactate dehydrogenase (LDH) were probed on the 10 ns to 10 ms time scale using laser-induced temperature-jump relaxation spectroscopy employing both UV fluorescence and isotope-edited IR absorption spectroscopy as structural probes. The dynamics of the mobile loop, which closes over the active site and is important for catalysis and binding, were characterized by studies of the inhibitor oxamate binding to the LDH/NADH binary complex monitoring the changes in emission of bound NADH. The bound NAD-pyruvate adduct, whose pyruvate moiety likely interacts with the same residues that interact with pyruvate in its ternary complex with LDH, served as a probe for any relative motions of active site residues against the substrate. The frequencies of its C=O stretch and -COO(-) antisymmetric stretch shift substantially should any relative motion of the polar moieties at the active site (His-195, Asp-168, Arg-109, and Arg-171) occur. The dynamics associated with loop closure are observed to involve several steps with motions from 1 to 300 microms. Apart from the "melting" of a few residues on the protein's surface, no kinetics were observed on any time scale in experiments of the bound NAD-pyr adduct although the measurements were made with a high degree of accuracy, even for final temperatures close to the unfolding transition of the protein. This is contrary to simple physical considerations and models. These results show that, once a productive protein/substrate complex is formed, the binding pocket is very rigid with very little, if any, motion apart from the mobile loop. The results also show that loop opening involves concomitant movement of the substrate out of the binding pocket.
- Published
- 2002
- Full Text
- View/download PDF
19. Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions.
- Author
-
Sauve AA, Celic I, Avalos J, Deng H, Boeke JD, and Schramm VL
- Subjects
- Adenosine Diphosphate Ribose chemistry, Adenosine Diphosphate Ribose metabolism, Amino Acid Sequence, Arabinose chemistry, Archaeal Proteins chemistry, Archaeal Proteins genetics, Bacterial Proteins chemistry, Bacterial Proteins genetics, Deuterium Oxide metabolism, Enzyme Inhibitors chemistry, Fungal Proteins chemistry, Fungal Proteins genetics, Histone Deacetylase Inhibitors, Histone Deacetylases genetics, Histone Deacetylases metabolism, Humans, Isomerism, Kinetics, Molecular Sequence Data, NAD metabolism, Nuclear Magnetic Resonance, Biomolecular, O-Acetyl-ADP-Ribose, Oxygen Isotopes metabolism, Sirtuin 1, Sirtuin 2, Sirtuins, Substrate Specificity, Trans-Activators antagonists & inhibitors, Trans-Activators genetics, Trans-Activators metabolism, Adenosine Diphosphate Ribose analogs & derivatives, Adenosine Diphosphate Ribose chemical synthesis, Gene Silencing, Histone Deacetylases chemistry, NAD chemistry, Silent Information Regulator Proteins, Saccharomyces cerevisiae, Trans-Activators chemistry
- Abstract
The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha-1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' --> 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.
- Published
- 2001
- Full Text
- View/download PDF
20. Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase.
- Author
-
Sharma R, Deng H, Leung A, and Mahuran D
- Subjects
- Amino Acid Sequence, Animals, Arginine genetics, Asparagine genetics, Binding Sites genetics, CHO Cells, Cricetinae, Disaccharides metabolism, Hexosaminidase B, Hexosaminidases genetics, Humans, Isoenzymes genetics, Kinetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Sequence Alignment, Substrate Specificity genetics, Transfection, beta-N-Acetylhexosaminidases genetics, Hexosaminidases metabolism, Isoenzymes metabolism, beta-N-Acetylhexosaminidases metabolism
- Abstract
In humans, beta-hexosaminidase A (alphabeta) is required to hydrolyze GM2 ganglioside. A deficiency of either the alpha- or beta-subunit leads to a severe neurological disease, Tay-Sachs or Sandhoff disease, respectively. In mammals beta-hexosaminidase B (betabeta) and S (alphaalpha) are other major and minor isozymes. The primary structures of the alpha- and beta-subunits are 60% identical, but only the alpha-containing isozymes can efficiently hydrolyze beta-linked GlcNAc-6-SO(4) from natural or artificial substrates. Hexosaminidase has been grouped with glycosidases in family 20. A molecular model of the active site of the human hexosaminidase has been generated from the crystal structure of a family 20 bacterial chitobiase. We now use the chitobiase structure to identify residues close to the carbon-6 oxygen of NAG-A, the nonreducing beta-GlcNAc residue of its bound substrate. The chitobiase side chains in the best interactive positions align with alpha-Asn(423)Arg(424) and beta-Asp(453)Leu(454). The change in charge from positive in alpha to negative in beta is consistent with the lower K(m) of hexosaminidase S, and the much higher K(m) and lower pH optimum of hexosaminidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesis, CHO cell expression, and kinetic analyses of an alphaArg(424)Lys hexosaminidase S detected little change in V(max) but a 2-fold increase in K(m) for the sulfated substrate. Its K(m) for the nonsulfated substrate was unaffected. When alphaAsn(423) was converted to Asp, again only the K(m) for the sulfated substrate was changed, increasing by 6-fold. Neutralization of the charge on alphaArg(424) by substituting Gln produced a hexosaminidase S with a K(m) decrease of 3-fold and a V(max) increased by 6-fold for the unsulfated substrate, parameters nearly identical to those of hexosaminidase B at pH 4.2. As well, for the sulfated substrate at pH 4.2 its K(m) was increased 9-fold and its V(max) decreased 1.5-fold, values very similar to those of hexosaminidase B obtained at pH 3.0, where its betaAsp(453) becomes protonated.
- Published
- 2001
- Full Text
- View/download PDF
21. Vibrational structure of GDP and GTP bound to RAS: an isotope-edited FTIR study.
- Author
-
Cheng H, Sukal S, Deng H, Leyh TS, and Callender R
- Subjects
- Electron Spin Resonance Spectroscopy, Guanosine Diphosphate metabolism, Guanosine Triphosphate metabolism, Humans, Ligands, Oxygen metabolism, Oxygen Isotopes metabolism, Protein Binding, Proto-Oncogene Proteins p21(ras) metabolism, Substrate Specificity, Guanosine Diphosphate chemistry, Guanosine Triphosphate chemistry, Proto-Oncogene Proteins p21(ras) chemistry, Spectroscopy, Fourier Transform Infrared methods
- Abstract
A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational modes. The symmetric modes of isotopically enriched enzyme-bound ligands can be obtained by Raman difference spectroscopy, but until now, the antisymmetric modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR difference spectroscopy, assign the antisymmetric modes, and accurately describe bonding. The method is used to assess the bonding changes that occur as Mg.GDP and Mg.GTP move from solution into the active site of RAS. Binding to RAS opens the nonbridging, O--P--O angle of the gamma-phosphate of GTP by 2.7 degrees, yet the angular freedom (dispersion of the O--P--O angle) of the gamma-phosphate is comparable to that in solution. In contrast, the motion of the beta-phosphate of GDP is highly restricted, suggesting that it positions the gamma-phosphate for nucleophilic attack. The beta,gamma-bridging O-P bond of bound GTP is slightly weakened, being lengthened by 0.005 A in the active site, corresponding to a bond order decrease of 0.012 valence unit (vu). The observed binding changes are consistent with a RAS-mediated hydrolysis mechanism that parallels that for solution hydrolysis.
- Published
- 2001
- Full Text
- View/download PDF
22. Dynamics of protein ligand binding on multiple time scales: NADH binding to lactate dehydrogenase.
- Author
-
Deng H, Zhadin N, and Callender R
- Subjects
- Animals, Binding Sites, Kinetics, Ligands, Models, Chemical, Niacinamide chemistry, Protein Binding, Spectrometry, Fluorescence methods, Static Electricity, Swine, Temperature, Thermodynamics, L-Lactate Dehydrogenase chemistry, L-Lactate Dehydrogenase metabolism, NAD chemistry, NAD metabolism, Proteins chemistry, Proteins metabolism
- Abstract
Although the importance of atomic motion to how proteins function has been conjectured for several decades, the characterization of protein dynamics on multiple time scales is scant. This is because of severe experimental and theoretical difficulties, particularly characterizing the nanosecond to millisecond time scales. Here, we apply advanced laser-induced temperature-jump relaxation spectroscopic techniques to examine the kinetics of NADH binding to lactate dehydrogenase over this time scale. The bimolecular rate process, at about 290 micros, is easily observed as are multiple faster events (with relaxation times of 200 ns, 3.5 micros, and 24 micros), revealing a rich dynamical nature of the binding step. The results show that there are multiple structures of bound enzyme-ligand complexes, some of which are likely to be far from the catalytically productive structure. The results have important implications for interpretations of the binding thermodynamics of ligands to LDH and, by extension, to other proteins. The observed processes likely play a role in the dynamics of the chemistry that is catalyzed by lactate dehydrogenase.
- Published
- 2001
- Full Text
- View/download PDF
23. Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization.
- Author
-
Deng HB, Yu Y, Pak Y, O'Dowd BF, George SR, Surratt CK, Uhl GR, and Wang JB
- Subjects
- Adenylyl Cyclases metabolism, Amino Acid Sequence, Animals, CHO Cells, Colforsin pharmacology, Cricetinae, Enkephalin, Ala(2)-MePhe(4)-Gly(5)- metabolism, Humans, Molecular Sequence Data, Mutation, Phosphorylation, Protein Binding genetics, Receptors, Opioid, mu chemistry, Transfection, Receptors, Opioid, mu genetics
- Abstract
Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.
- Published
- 2000
- Full Text
- View/download PDF
24. A Raman spectroscopic characterization of bonding in the complex of horse liver alcohol dehydrogenase with NADH and N-cyclohexylformamide.
- Author
-
Deng H, Schindler JF, Berst KB, Plapp BV, and Callender R
- Subjects
- Alcohol Dehydrogenase antagonists & inhibitors, Animals, Horses, Hydrogen Bonding, Kinetics, Macromolecular Substances, Magnetic Resonance Spectroscopy, Mathematical Computing, Models, Chemical, Protein Binding, Spectrum Analysis, Raman methods, Thermodynamics, Alcohol Dehydrogenase chemistry, Formamides chemistry, Liver enzymology, NAD chemistry
- Abstract
The binding of N-cyclohexylformamide (CXF) to the complex of horse liver alcohol dehydrogenase with NADH mimics that of the Michaelis complex for aldehyde reduction catalyzed by the enzyme. The Raman spectra of bound CXF and its 13C- and 15N-substituted derivatives have been obtained using Raman difference techniques, and the results are compared with CXF spectra in aqueous solution and in methylene chloride. The results indicate that the amide N-H bond is trans to the C=O bond of CXF both in solution and in the enzyme ternary complex. The C=O stretch and N-H bending modes of the amide of CXF shift -16 and -9 cm-1, respectively, in the enzyme ternary complex relative to that in aqueous solution and -48 and 36 cm-1, respectively, relative to that in methylene chloride. Ab initio normal mode calculations on various model systems of CXF show that the observed frequency changes of the C=O stretch mode have contributions from the frequency changes induced by the environmental changes near both the local C=O bond and the remote N-H bond. The same is true for the observed N-H bending frequency change. Our calculations also show that the environmentally induced frequency changes are additive so that it is possible to determine the C=O stretch (or N-H bending) frequency change that is due to the local interaction change near the C=O (or N-H) bond from the observed frequency changes. On the basis of these results and the empirical relationship between the C=O stretch frequency shift and the interaction enthalpy change on the C=O bond developed here, it is found that the C=O group of CXF in the enzyme/NADH/CXF complex binds with a favorable interaction enthalpy of approximately 5.5 kcal/mol relative to water. Similar analysis suggests that the N-H moiety of CXF is destabilized in the ternary complex by about 1.5 kcal/mol relative to water but is stabilized by about 1.5 kcal/mol relative to a hydrophobic environment. The analysis describes quantitatively the binding of the C=O of CXF with the catalytic zinc and the hydroxyl group of Ser-48 and the interaction of the N-H with the benzene ring of Phe-93 of the enzyme.
- Published
- 1998
- Full Text
- View/download PDF
25. Raman difference spectroscopic studies of the myosin S1.MgADP.vanadate complex.
- Author
-
Deng H, Wang J, Callender RH, Grammer JC, and Yount RG
- Subjects
- Adenosine Diphosphate metabolism, Animals, Anions, Binding Sites, Catalysis, Hydrolysis, Macromolecular Substances, Models, Chemical, Myosin Subfragments metabolism, Rabbits, Solutions, Spectrum Analysis, Raman, Vanadates metabolism, Adenosine Diphosphate chemistry, Myosin Subfragments chemistry, Vanadates chemistry
- Abstract
The Raman spectra of the nonbridging V--O bonds in the myosin S1.MgADP.Vi complex, often believed to be a transition-state analogue for the phosphotransfer reaction catalyzed by myosin, and in a vanadate solution model compound have been obtained using Raman difference spectroscopic techniques. A symmetric/asymmetric pair of modes at 870 cm-1 is found for vanadate in solution while three bands are found in the myosin S1.MgADP.Vi complex at 870, 844, and 829 cm-1. Using empirical relationships that relate bond order/bond lengths to stretch frequencies, the bond order and bond length of the three nonbridging V--O bonds of vanadate in solution were determined to be 1.43 vu (+/-0.04 vu) and 1.669 A (+/-0.004 A), respectively. The average bond order and bond length of the nonbridging V--O bonds in the S1.MgADP.Vi complex were determined to be 1.38 vu and 1.683 A. A normal-mode analysis suggests that the VO32- moiety approaches a planar conformation in the enzymic complex. Ab initio calculations show that a water molecule at the S1 ATPase binding site, in line with the apical O-V bond in the ADP-Vi moiety and believed to be the attacking nucleophile in the phosphotransfer reaction, can account well for the changes in frequencies of vanadate when it binds to the protein by forming a moderately strong V-O(H2) bond. Hence, an important role determining the ATPase activity at the active site of myosin appears to be a strategic positioning of this in-line water molecule. Assuming that the distortions that vanadate undergoes upon forming the S1.MgADP.Vi complex are analogous to the changes of the gamma-phosphate of ATP in the transition state of the myosin-catalyzed hydrolysis, our results suggest that this reaction proceeds close to a concerted (SN2-like) process.
- Published
- 1998
- Full Text
- View/download PDF
26. Characterization of hydrogen bonding in the complex of adenosine deaminase with a transition state analogue: a Raman spectroscopic study.
- Author
-
Deng H, Kurz LC, Rudolph FB, and Callender R
- Subjects
- Adenosine Deaminase metabolism, Animals, Catalysis, Hydrogen Bonding, Mice, Purine Nucleosides chemistry, Ribonucleosides chemistry, Spectrum Analysis, Raman, Adenosine Deaminase chemistry
- Abstract
The Raman spectra of purine ribonucleoside as well as a stable model compound (1-methoxyl-1,6-dihydropurine ribonucleoside), free in solution and bound into its complex with adenosine deaminase (ADA), have been studied by Raman difference spectroscopy. Using purine riboside analogues labeled with 15N1 or 13C6 and the theoretical frequency normal-mode analyses of these molecules using ab initio quantum mechanic methods, we have positively identified many of the Raman bands in the enzyme-bound inhibitor. The spectrum of the enzyme-bound inhibitor is consistent with the enzyme-catalyzed hydration of the purine base to yield 1-hydroxyl-1,6-dihydropurine ribonucleoside, as suggested earlier by X-ray crystallographic studies. In addition, the Raman data and subsequent vibrational analyses show that the binding-induced Raman spectral changes of the inhibitor can be modeled by the formation of a strong hydrogen bond to its N1-H bond. This hydrogen bond, apparently between the N1-H of the inhibitor and the Odelta1 of Glu217 in ADA, causes a substantial N1-H bending frequency increase of about 50-100 cm-1 compared to its solution value, and this results in an estimated enthalpy of the hydrogen bond of 4-10 kcal/mol. The relationship of transition state stabilization in the catalytic strategy of this efficient enzyme to such a bonding pattern is discussed.
- Published
- 1998
- Full Text
- View/download PDF
27. Biochemical characterization of the Cys138Arg substitution associated with the AB variant form of GM2 gangliosidosis: evidence that Cys138 is required for the recognition of the GM2 activator/GM2 ganglioside complex by beta-hexosaminidase A.
- Author
-
Xie B, Rigat B, Smiljanic-Georgijev N, Deng H, and Mahuran D
- Subjects
- Amino Acid Sequence, Animals, CHO Cells, Cricetinae, Cysteine physiology, Disulfides, Escherichia coli genetics, G(M2) Activator Protein, Gangliosidoses enzymology, Genetic Variation, Molecular Sequence Data, Oligopeptides, Peptides genetics, Protein Folding, Proteins genetics, Recombinant Fusion Proteins biosynthesis, Amino Acid Substitution genetics, Arginine genetics, Cysteine genetics, G(M2) Ganglioside genetics, Gangliosidoses genetics, Proteins metabolism, beta-N-Acetylhexosaminidases metabolism
- Abstract
The function of the GM2 activator protein is to act as a substrate-specific cofactor in the hydrolysis of GM2 ganglioside by beta-hexosaminidase A. Mutations in the gene encoding it result in the AB variant form of GM2 gangliosidosis. One such mutation, Cys138 Arg, results in the mutant protein being retained and degraded in the endoplasmic reticulum of mammalian cells. In order to characterize the biochemical effects of this substitution, we expressed the mutant protein in transformed bacteria. We first compared the wild-type protein produced by two bacterial expression methods, one requiring protein refolding, with activator purified from the medium of transfected CHO cells. The "activity" and circular dichroism spectrum (alpha-helical content) of all three proteins were similar, justifying the use of refolded activator from transformed bacteria in structure/function studies. Second, the mutant protein was expressed in both bacterial systems and in each retained approximately 2% of the wild type's specific activity. The presence of even this small amount of activity in the mutant protein coupled with a calculated alpha-helical content nearly identical to the wild type, strongly suggest that no major tertiary or secondary structural changes, respectively, had occurred due to the mutation. However, we demonstrate that its heat stability at 60 degrees C is reduced 14-fold, suggesting some localized change in tertiary structure. The loss of a disulfide loop was confirmed by reacting the mutant protein with Ellman's reagent. A kinetic analysis detected a large increase in the apparent K(m) of beta-hexosaminidase A for the mutant; however, there was no apparent change in Vmax. A fluorescence dequenching assay was used to evaluate the ability of the mutant protein to transport lipids and bind GM2 ganglioside. These assays detected no difference between the wild-type and mutant proteins, indicating that the Cys138 Arg substitution has no effect on these functions. We conclude that the mutation specifically affects a domain in the activator protein that is responsible for the recognition of the activator-GM2 ganglioside complex by beta-hexosaminidase A.
- Published
- 1998
- Full Text
- View/download PDF
28. Importance of the gamma-carboxyl group of glutamate-462 of the large alpha-subunit for the catalytic function and the stability of the multienzyme complex of fatty acid oxidation from Escherichia coli.
- Author
-
He XY, Deng H, and Yang SY
- Subjects
- 3-Hydroxyacyl CoA Dehydrogenases metabolism, Acetyl-CoA C-Acyltransferase metabolism, Binding Sites genetics, DNA Primers, Enoyl-CoA Hydratase metabolism, Escherichia coli chemistry, Escherichia coli genetics, Gene Expression genetics, Glutamic Acid chemistry, Glutamic Acid metabolism, Histidine chemistry, Histidine metabolism, Hydrogen-Ion Concentration, Isomerases metabolism, Kinetics, Mutagenesis, Site-Directed, Mutation genetics, Protein Conformation, Protein Denaturation, Racemases and Epimerases metabolism, Temperature, 3-Hydroxyacyl CoA Dehydrogenases chemistry, Acetyl-CoA C-Acyltransferase chemistry, Carbon-Carbon Double Bond Isomerases, Enoyl-CoA Hydratase chemistry, Enzyme Stability, Escherichia coli enzymology, Isomerases chemistry, Racemases and Epimerases chemistry
- Abstract
His450 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was recently identified as an essential catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase [He, X-Y., & Yang, S.-Y. (1996) Biochemistry 35, 9625-9630]. To explore the roles of acidic residues in the dehydrogenase catalysis, every conserved acidic residue in the dehydrogenase functional domain except for those in the NAD-binding motif was replaced with alanine. The resulting mutant complexes were overproduced and characterized. Their component enzymes other than the dehydrogenase were affected very slightly. Removal of the beta-carboxyl group of Asp524 and Asp542 caused only a 3- and 4-fold, respectively, decrease in the catalytic efficiency of the dehydrogenase, thereby showing that their involvement in the dehydrogenase catalysis was limited. In contrast, the alpha/Glu462-->Ala mutant complex showed a greater than 160-fold reduction in the kcat of the dehydrogenase in the forward direction without a significant change of the k(m) for the substrate. The catalytic properties of the alpha/Glu462-->Gln mutant complex were found to be similar to those of the alpha/Glu462-->Ala mutant complex except that the kcat of the dehydrogenase in the backward direction was about 4-fold lower and the Km for the substrate of the thiolase was 6-fold higher. It is concluded that the negative charge of the gamma-carboxyl group of Glu462, but not its ability to form a hydrogen bond, is critical for its interaction with His450, thereby assisting in the catalysis of the dehydrogenase. The pKa of His450 in the E.NADH binary complex was virtually unchanged by the replacement of Glu462 with Ala or Gln. It seems that the binding of substrate is necessary for forming a strong interaction between His450 and Glu462 with the result that the electroneutrality in the active site is maintained and the activation energy of the reaction is lowered. Additionally, the negative charge of Glu462 increases the thermostability of the multienzyme complex.
- Published
- 1997
- Full Text
- View/download PDF
29. Trypanosomal nucleoside hydrolase. Resonance Raman spectroscopy of a transition-state inhibitor complex.
- Author
-
Deng H, Chan AW, Bagdassarian CK, Estupiñán B, Ganem B, Callender RH, and Schramm VL
- Subjects
- Animals, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacology, Models, Molecular, N-Glycosyl Hydrolases antagonists & inhibitors, N-Glycosyl Hydrolases metabolism, Protein Conformation, Pyrroles metabolism, Pyrroles urine, Spectrum Analysis, Raman, N-Glycosyl Hydrolases chemistry, Trypanosoma enzymology
- Abstract
The transition state for hydrolysis of the N-ribosidic bond of inosine by nucleoside hydrolase has oxocarbenium character and a protonated leaving group hypoxanthine with an sp2-hybridized C1' of the ribosyl [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. These features are incorporated into N-(p-nitrophenyl)-D-riboamidrazone, a transition state analogue which binds with a dissociation constant of 2 nM [Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V. L., & Ganem, B. (1994) Biochemistry 33, 3994-4000]. Resonance Raman and ultraviolet-visible absorbance spectroscopy has established that the inhibitor binds as the neutral, zwitterionic species. The enzyme stabilizes a specific resonance state characterized by the quinonoid form of the p-nitrophenyl group with evidence for ion pairing at the nitro group. Incorporation of 15N into a specific position of the amidrazone reveals that the exo-ribosyl nitrogen bonded to the C1' position carries the proton while that bonded to the p-nitrophenyl carbon is unprotonated. This tautomer carries a distributed positive charge centered at the position analogous to C1' of the ribosyl group at the transition state. The molecular electrostatic potentials for the substrate inosine, the transition state, and the transition state inhibitor are compared at the van der Waals surface of the molecules. The tautomer of the inhibitor bound to the enzyme bears a striking electrostatic resemblance to the transition state determined by kinetic isotope effect analysis. The spectral and resonance Raman properties of free and enzyme-bound inhibitor have permitted tautomeric assignment of these species and establish that the enzyme substantially changes the electronic distribution of the bound inhibitor toward that of the enzyme-stabilized transition state.
- Published
- 1996
- Full Text
- View/download PDF
30. Chiral recognition of deoxyoligonucleotides by delta- and lambda-tris(ethylenediamine)cobalt(III).
- Author
-
Xu Q, Jampani SR, Deng H, and Braunlin WH
- Subjects
- Base Sequence, Circular Dichroism, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Oligodeoxyribonucleotides chemistry, Salts, Solutions, Stereoisomerism, Cobalt chemistry, DNA chemistry, Ethylenediamines chemistry, Organometallic Compounds chemistry
- Abstract
59Co NMR and CD measurements show for both stereoisomers of Co(en)3(3+) a similar trend in the sequence dependence of DNA recognition, as was reported previously for Co(NH3)6(3+). In particular, specific binding is evident to DNA molecules possessing runs of two or more same-strand guanine residues. The binding of either isomer to such sequences induces structural transitions toward A-DNA characteristics. Such measurements also show significant differences between the two stereoisomers in terms of how they recognize specific duplex DNA sequences. delta-Co(en)3(3+) binds more tightly than lambda-Co(en)3(3+) to right-handed, guanine-rich DNA, whereas lambda-Co(en)3(3+) binds more tightly than delta-Co(en)3(3+) to left-handed DNA. The two stereoisomers bind in an indistinguishable manner to AT-rich DNA.
- Published
- 1995
- Full Text
- View/download PDF
31. Source of catalysis in the lactate dehydrogenase system. Ground-state interactions in the enzyme-substrate complex.
- Author
-
Deng H, Zheng J, Clarke A, Holbrook JJ, Callender R, and Burgner JW 2nd
- Subjects
- Animals, Catalysis, Geobacillus stearothermophilus enzymology, NAD analogs & derivatives, Pyruvates chemistry, Pyruvic Acid, Spectrum Analysis, Raman, Substrate Specificity, Swine, Thermodynamics, L-Lactate Dehydrogenase metabolism
- Abstract
The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C(+)-O- charge separation that was seen previously with the mammalian enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C = O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C V O and the protonated His-195. Thus, while the C = O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stability substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
- Full Text
- View/download PDF
32. Internal chemical bonding in solutions of simple phosphates and vanadates.
- Author
-
Ray WJ Jr, Burgner JW 2nd, Deng H, and Callender R
- Subjects
- Magnetic Resonance Spectroscopy, Oxygen chemistry, Spectrum Analysis, Raman, Phosphates chemistry, Vanadates chemistry
- Abstract
The chemical bonding within structurally related phosphates and vanadates in aqueous solution is compared on the basis of vibrational frequencies obtained by classical Raman spectroscopy. To do this, an empirical relationship between the stretching frequency of P-O and P-OH or P-OR groups and bond strength is developed such that the sum of the PO bond strengths, expressed in terms of average number of electron pairs per bond, is as close as possible to 5.0 for phosphoric acid and various anions and esters thereof. The same approach is used for the corresponding vanadates. The internal bonding in phosphates involves a greater bond strength for P-OH and a smaller strength for P-O than might be expected from a simple consideration of canonical resonance forms. In vanadates, V-OH and V-O are closer to single and double bonds, respectively, than in phosphates, and the force constant for V = O is considerably smaller than for P = O, although that for V-OH and P-OH is similar. Since treating the P-O and V-O groups of simple tetrahedral phosphates and vanadates as independent diatomic oscillators provides good correlations between the respective frequencies and bond strengths, the same correlations are used to approximate the expected stretching frequencies for distorted phosphates and vanadates. The distortions considered are those that presumably characterize associative and dissociative transition states for a concerted transfer of the (PO3-) fragment of a dianionic phosphate group between donor and acceptor oxygens with similar character.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
- Full Text
- View/download PDF
33. Molecular cloning and chromosomal assignment of the gene for human Zn-alpha 2-glycoprotein.
- Author
-
Ueyama H, Deng HX, and Ohkubo I
- Subjects
- Amino Acid Sequence, Base Sequence, Chromosome Mapping, Cloning, Molecular, Genes, Genomic Library, Humans, In Situ Hybridization, Fluorescence, Molecular Sequence Data, Pseudogenes, Sequence Alignment, Sequence Homology, Nucleic Acid, Zn-Alpha-2-Glycoprotein, Chromosomes, Human, Pair 7, Glycoproteins genetics, Seminal Plasma Proteins
- Abstract
Genomic clones containing the human Zn-alpha 2-glycoprotein gene were isolated. Two of them were for the functional gene overlapped, and the other four were for two different pseudogenes (1 and 2) retaining exon-intron organization. The complete DNA sequence of the functional gene (9.3 kb) and its 5'- and 3'-flanking regions (5.3 and 0.1 kb, respectively) was determined. The gene is composed of four exons; the first exon is for the 5'-untranslated region, the signal sequence, and the first six amino acids; the second, for domain A; the third, for domain B; and the fourth, for domain C and the 3'-untranslated region. The 5'-flanking region contains a TATA box, a CAT box, an octamer sequence, and three possible Spl-binding sites. Ten and three copies of Alu repetitive DNA were identified within the gene and the 5'-flanking region, respectively, and they occupy 30% of the gene. The nucleotide sequences around the exons of pseudogene 1 were also determined; they had high homology (90-91%) with the corresponding region of the functional gene. Southern blot analysis suggested that there are only three genes, including nonfunctional ones, for Zn-alpha 2-glycoprotein in humans. The gene (ZA2G) was mapped to human chromosome band 7q22.1 by fluorescence in situ hybridization.
- Published
- 1993
- Full Text
- View/download PDF
34. Selective localization and rotational immobilization of univalent cations on quadruplex DNA.
- Author
-
Xu Q, Deng H, and Braunlin WH
- Subjects
- Cations, Monovalent, Magnetic Resonance Spectroscopy, Motion, Nucleic Acid Conformation, Oligodeoxyribonucleotides chemistry, Potassium chemistry, Sodium chemistry, DNA chemistry
- Abstract
The quadruplex structure of the oligomer d(T2G4T) is more stable in the presence of K+ than in the presence of Na+. This enhanced stability correlates with the preferential binding of K+ to a small number of specific sites on the quadruplex. In contrast, Na+ and K+ compete on an equal footing for atmospheric binding. Both 39K+ and 23Na+ are, when specifically bound, significantly inhibited in their rotational mobility, so that the quadrupolar relaxation reflects the molecular tumbling of the oligomer, which occurs on the time scale of nanoseconds. This rotational immobilization is in distinct contrast to the high rotational mobility of atmospherically bound cations. On the other hand, all NMR-visible 39K+ in solution is in rapid exchange among all environments (free, specifically bound, and atmospherically bound) implying that the lifetime of specifically coordinated 39K+ must be significantly shorter than a millisecond. A similar conclusion holds for 23Na+. The oligomer d(T2G4T) forms two distinct Hoogsteen base-paired structures in NaCl solution, separated by a large kinetic barrier. Neither of these structures is as stable with respect to base pair opening as is the quadruplex structure formed in KCl solution. Only one of these two structures is associated with rotational immobilization of bound 23Na+.
- Published
- 1993
- Full Text
- View/download PDF
35. Comparison of vibrational frequencies of critical bonds in ground-state complexes and in a vanadate-based transition-state analog complex of muscle phosphoglucomutase. Mechanistic implications.
- Author
-
Deng H, Ray WJ Jr, Burgner JW 2nd, and Callender R
- Subjects
- Animals, Binding Sites, Cations, Divalent chemistry, Glucosephosphates chemistry, Glucosephosphates metabolism, In Vitro Techniques, Muscles enzymology, Phosphates metabolism, Rabbits, Spectrum Analysis, Raman, Vanadates chemistry, Phosphoglucomutase chemistry
- Abstract
The symmetric stretching frequency of the P-O bonds of the enzymic phosphate group in muscle phosphoglucomutase was measured via 16O/18O Raman difference spectroscopy. This frequency, and its shift on isotopic substitution, is characteristic of a dianionic phosphate ester. The P-O stretching frequency is not detectably altered by the binding of the metal ion activators Mg2+, Zn2+, or Cd2+ nor by the subsequent binding of glucose phosphate. Hence, a binding-induced distortion/polarization of the enzymic phosphate group in the ground state, or enzyme-substrate complex, cannot serve as a rationale for the large value of kcat in the phosphoglucomutase reaction. By contrast, the stretching frequency of the V-O bonds within a vanadate group bound at the same site in the transition-state analog complex involving glucose 1-phosphate 6-vanadate is much lower than for a normal dianionic vanadate. This low V-O stretching frequency is best rationalized in terms of the extensive polarization of all three nonbridging oxygens of the vanadate ester dianion plus the formation of a weak, fifth bond to the vanadium atom. This distortion/polarization of the VO3(2-) group depends on the metal ion activator, since it is largely abolished, and the involvement of the fifth ligand eliminated, by substitution of Li+ for Mg2+ at the metal activation site.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
- Full Text
- View/download PDF
36. Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy.
- Author
-
Goldberg JM, Zheng J, Deng H, Chen YQ, Callender R, and Kirsch JF
- Subjects
- Aspartate Aminotransferases genetics, Aspartate Aminotransferases metabolism, Chemical Phenomena, Chemistry, Physical, Hydrogen Bonding, Molecular Structure, Mutation, Pyridoxal Phosphate metabolism, Schiff Bases chemistry, Spectrophotometry, Ultraviolet, Aspartate Aminotransferases chemistry, Escherichia coli enzymology, Phenylalanine chemistry, Pyridoxal Phosphate chemistry, Spectrum Analysis, Raman, Tyrosine chemistry
- Abstract
The azomethine (Schiff base) linkage between the epsilon-amino group of active-site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'-phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (lambda max = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H(2)18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H(2)18O.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
- Full Text
- View/download PDF
37. Catalytic RNA reactions of yeast tRNA(Phe) fragments.
- Author
-
Deng HY and Termini J
- Subjects
- Base Sequence, Circular Dichroism, Kinetics, Lead metabolism, Molecular Sequence Data, Nucleic Acid Conformation, RNA, Catalytic chemistry, RNA, Fungal chemistry, RNA, Transfer, Phe chemistry, Ribonuclease H metabolism, Temperature, RNA, Catalytic metabolism, RNA, Fungal metabolism, RNA, Transfer, Phe metabolism, Saccharomyces cerevisiae genetics
- Abstract
We describe 12 new catalytic RNA reactions which are intermolecular variants of the well-known intramolecular Pb(2+)-promoted hydrolysis of yeast tRNA(Phe). Fragments derived from the native yeast tRNA(Phe) which possess the T stem-loop can function as catalysts for the site-specific hydrolysis at p18 of D stem-loop-containing fragments. An initial report described the catalytic cleavage of an unmodified T7 transcript corresponding to the 5' half of tRNA(Phe) by a 3' half-molecule derived from the native tRNA. [Sampson, J. R., Sullivan, F. X., Behlen, L. S., DiRenzo, A. B., & Uhlenbeck, O. C. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 267-275]. We have investigated the trans reaction further by creating a family of substrate and catalyst RNA molecules by dissection of the native tRNA(Phe) using a combination of chemical and enzymatic methods. A search for cleavage activity in trans was conducted using a combinatorial approach with the available T and D stem-loop-containing fragments. Twelve combinations were found to be catalytic, and initial rates, kcat's, and Km's are reported for each. The kcat's for the reactions differ by approximately 20-fold, whereas Km's vary by only approximately 2-fold. Differences in some of the cleavage rates argue that tertiary interactions present in the intact molecule can be reconstituted in the fragment combinations. Secondary structural features remote from the cleavage site can also affect the apparent cleavage rates. A minimum catalytic complex consisting of a substrate fragment corresponding to nucleotides 1-24 of the native molecule and a catalytic RNA corresponding to 46-76 is identified. This complex is of interest since the transition state for cleavage involves only three helices, with no elements of the anticodon required for cleavage. This is reminiscent of the proposed secondary structure of the hammerhead catalytic RNA cleavage motif.
- Published
- 1992
- Full Text
- View/download PDF
38. A vibrational analysis of the catalytically important C4-H bonds of NADH bound to lactate or malate dehydrogenase: ground-state effects.
- Author
-
Deng H, Zheng J, Sloan D, Burgner J, and Callender R
- Subjects
- Animals, Carbon chemistry, Catalysis, Mitochondria enzymology, Myocardium enzymology, NAD metabolism, Spectrum Analysis, Raman, Swine, L-Lactate Dehydrogenase metabolism, Malate Dehydrogenase metabolism, NAD chemistry
- Abstract
We have measured the frequency of the carbon-hydrogen stretching mode of the pro-R and pro-S C4-H bonds of NADH in solution and when bound to pig heart lactate (LDH) or mitochondrial malate (mMDH) dehydrogenases. This is achieved by specifically deuterating the C4 pro-R or pro-S hydrogens of NADH and determining the frequencies of the resulting C4-D stretches by Raman difference spectroscopy. We find that the frequencies of the two C4-D stretching modes for the two bonds are essentially the same for the unliganded coenzyme. On the other hand, the position of the pro-S-[4-2H]NADH stretch shifts upward by about 23-30 cm-1 in its binary complex with either lactate or malate dehydrogenase relative to that observed in solution, while that for the bound pro-R-[4-2H]NADH is relatively unchanged. The fact that the frequency of the pro-R hydrogen is not significantly affected during complex formation suggests that the rate enhancements for reaction of substrate with NADH brought about by both pig heart LDH and mMDH apparently do not involve either stabilization or destabilization of the pro-R hydrogen of NADH in enzyme-coenzyme binary complexes, in agreement with previous chemical studies. That these proteins are able to regulate the frequencies of the two C4-D bonds differentially, and hence the electronic distributions in these bonds, has important implications for the stereochemical reactions catalyzed by the NAD dehydrogenases, and this is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1992
- Full Text
- View/download PDF
39. An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21.
- Author
-
Manor D, Weng GZ, Deng H, Cosloy S, Chen CX, Balogh-Nair V, Delaria K, Jurnak F, and Callender R
- Subjects
- Amino Acid Sequence, Deuterium, Kinetics, Peptide Elongation Factor Tu chemistry, Proto-Oncogene Proteins p21(ras) chemistry, Radioisotope Dilution Technique, Spectrum Analysis, Raman methods, Tritium, Escherichia coli metabolism, Guanosine Diphosphate metabolism, Peptide Elongation Factor Tu metabolism, Proto-Oncogene Proteins p21(ras) metabolism
- Abstract
We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.
- Published
- 1991
- Full Text
- View/download PDF
40. Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques.
- Author
-
Deng H, Burgner J, and Callender R
- Subjects
- Cytoplasm chemistry, Cytoplasm enzymology, L-Lactate Dehydrogenase chemistry, Mitochondria chemistry, Mitochondria enzymology, Protein Binding, Spectrum Analysis, Raman, Coenzymes chemistry, Malate Dehydrogenase chemistry, NAD chemistry
- Abstract
We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5'-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide-NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1991
- Full Text
- View/download PDF
41. Resonance Raman studies of the HOOP modes in octopus bathorhodopsin with deuterium-labeled retinal chromophores.
- Author
-
Deng H, Manor D, Weng G, Rath P, Koutalos Y, Ebrey T, Gebhard R, Lugtenburg J, Tsuda M, and Callender RH
- Subjects
- Animals, Cattle, Deuterium, Hydrogen chemistry, Octopodiformes, Rhodopsin chemistry, Spectrum Analysis, Raman, Thermodynamics, Vibration, Rhodopsin analogs & derivatives
- Abstract
Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm-1 for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm-1 to C10H and C14H HOOP modes, and the band at 940 cm-1 to C11H = C12H Au-like HOOP mode. Except for a 26 cm-1 downward shift, the C11H = C12H Au-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C10H and C14H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C7H and C8H HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C11H = C12H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C11 = C12 double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C11H = C12H Au-like HOOP mode. The twisted nature of the chromophore, semiquantitatively discussed here, likely affects the lambda max of the chromophore and its enthalpy.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1991
- Full Text
- View/download PDF
42. Binding of S21 to the 50S subunit and the effect of the 50S subunit on nonradiative energy transfer between the 3' end of 16S RNA and S21.
- Author
-
Odom OW, Deng HY, Dabbs ER, and Hardesty B
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, Fluorescence Polarization, Macromolecular Substances, Molecular Weight, Mutation, Peptide Initiation Factors metabolism, RNA, Transfer, Amino Acyl metabolism, Ribosomal Proteins metabolism, RNA, Ribosomal metabolism, Ribosomes metabolism
- Abstract
Escherichia coli ribosomal protein S21 was labeled at its single cysteine group with a fluorescent probe. Labeled S21 showed full activity in supporting MS2 RNA-dependent binding of formylmethionyl-tRNAf to 30S ribosomal subunits. Fluorescence anisotropy measurements and direct analysis on glycerol gradients demonstrate conclusively that labeled S21 binds to 50S ribosomal subunits as well as to 30S and 70S particles. The relative binding affinities are in the order 70S greater than 30S greater than 50S. Other results presented appear to indicate that S21 is bound in the same position on either 50S subunits or 30S subunits as in 70S ribosomes, suggesting that the protein is bound simultaneously to both subunits in the latter. Addition of 50S subunits to 30S particles containing probes on S21 and at the 3' end of 16S RNA caused a decrease in the energy transfer between these points. The results correspond to an apparent change in distance from 51 to 61 A.
- Published
- 1984
- Full Text
- View/download PDF
43. Classical Raman spectroscopic studies of NADH and NAD+ bound to lactate dehydrogenase by difference techniques.
- Author
-
Deng H, Zheng J, Sloan D, Burgner J, and Callender R
- Subjects
- Animals, Hydrogen Bonding, Oxidation-Reduction, Protein Binding, Protein Conformation, Solvents, Spectrum Analysis, Raman methods, Swine, L-Lactate Dehydrogenase metabolism, NAD metabolism
- Abstract
The binding of the coenzymes NAD+ and NADH to lactate dehydrogenase causes significant changes in the Raman spectra of both of these molecules relative to spectra obtained in the absence of enzyme. The molecular motions of the bound adenine moiety of both NAD+ and NADH as well as adenine containing analogues of these coenzymes produce Raman bands that are essentially identical, suggesting that the binding of adenine to the enzyme is the same regardless of the nicotinamide head-group nature. We also have observed that the molecular motions of the bound adenine moiety are different from both those obtained when it is in either water, various hydrophobic solvents, or various other solvent compositions. Protonation of the bound adenine ring at the 3-position is offered as a possible explanation. Significant shifts are observed in both the stretching frequency of the carboxamide carbonyl of NAD+ and the rocking motion of the carboxamide NH2 group of NADH. These shifts are probably caused by hydrogen bonding with the enzyme. The interaction energies of these hydrogen-bonding patterns are discussed. The aromatic nature of the nicotinamide moiety of NAD+ appears to be unchanged upon binding. Pronounced changes in the Raman spectrum of the nicotinamide moiety of NADH are observed upon binding; some of these changes are understood and discussed. Finally, these results are compared to analogous results that were recently reported for liver alcohol dehydrogenase [Chen et al. (1987) Biochemistry 26, 4776-4784]. In general, the coenzyme binding properties are found to be quite similar, but not identical, for the two enzymes.
- Published
- 1989
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.