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Heteronuclear multidimensional nuclear magnetic resonance methods were used in the investigation of the structure of Delta5-3-ketosteroid isomerase homodimer complexed with 19-nortestosterone hemisuccinate in solution. Results reveal that each monomer of the enzyme consists of three alpha-helices, eight beta-strands and two beta-bulges. The three alpha-helices are situated at the concavity formed by the beta-sheet.

Low-temperature, high-field protein nuclear magnetic resonance spectroscopic studies were utilized to analyze the role of low-barrier hydrogen bond (LBHB) in the catalytic activity of triophosphate isomerase. A highly deshielded resonance was assigned to a LBHB from the NOH proton of bound phosphoglycolohydroxamic acid (PGH) to the general-base catalyst Glu-165. The strong interaction of PGH with Glu-165 indicated the proton transfer between the oxygen molecules and C1-C2 carbons.

The influence of Asp-99 and Tyr-14 in the catalysis of ketosteroid isomerase was determined through the use of 3D 1H-13C NOESY-HSQC spectrum of Tyr-14 mutant isomerase and UV resonance Raman spectroscopy. The H-bond donation by Tyr-14 was compensated by another H-bond donor. The side chain of Asp-99 was characterized to be proximal to the aromaric ring of Tyr-14. Asp-99 donation of H-bond was stronger that that of Tyr-14, confirming the importance of Asp and Tyr residues in the formation of H-bonds on enzymes.

Rapidly exchanging (greater than 1.0s-super1) backbone amide protons exchange rates with solvent water in staphylococcal nuclease (SN) were measured at pH 6.03-7.03 with a 2D heteronuclear water exchange filter sequence. It was found that non-hydrogen-bonding and exposed residues had protection factors less than 15 for non-hydrophobic residues. Protection factors for buried and non-hydrogen-bonded protons were found to vary, indicating kinetically labile hydrogen bonds and solvent exposure by structural fluctuations.

The secondary structure, in solution, of the Y55F/Y88F highly active mutant of isomerase, when complexed with 19-nortestosterone hemisuccinate, has been determined. The results of heteronuclear NMR studies showed two alpha-helices, one 3(sub 10) helix, seven beta-strands, and five turns. The isomerase, therefore, consists of 30% helix, 38% beta-sheet and 16% turns. The remaining 20 residues (16 %) are assumed to form coils.

The solution structure of the quaternary MutT-M2+-AMPCPP-M2+ complex was determined by employing AMPCPP which was previously proven to be a nonhydrolyzable substrate analog for MutT. Findings indicated that the binding of the essential divalent cation activator Mg2+ and the substrate analog Mg2+AMPCPP to the MutT enzyme to produce the quaternary E-Mg2+-AMPCPP-Mg2+ complex does not change the global fold of the enzyme but instead, creates localized, small conformational changes at or near the metal and substrate binding sites.

The conformational dynamics of the secondary structure of 4-oxalocrotonate tautomerase (4-OT) was analyzed by nuclear magnetic resonance spectroscopy. The backbone motion of the 41 kDa homohexamer free enzymes was restricted in the eight residues and six active site residues after binding with cis,cis-muconate. Large increases in the order of parameters was not detected in the active sites of the 4-OT backbone. However, the C-terminal coil of the enzyme exhibited a uniform increase in the order of parameter after binding with cis,cis-muconate.

[[Delta].sup.5]-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of [10.sup.10]. Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4[Beta]-proton to the 6[Beta]-position, contribute [10.sup.4.7] and [10.sup.5.6] to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the p[K.sub.a] values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, [Phi] = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.

The glutamate amino acid at the 57th position (Glu-57) in the Escherichia coli MutT enzyme functions as a ligand for the metal ion in MutT. The replacement of Glu-57 by a Gln residue decreases the activity of MutT, and the binding affinity for Mg2+ and Mn2+. However, the affinity for dGTP is unaffected by the mutation. The loss of enzymatic activity is due to electronic and structural alterations at the enzyme's active site. Mutations or neutralization that remove the negative charge at Glu-57 produce similar changes in the enzymatic activity.

The side chain and backbone dynamics of the catalytic residue, Tyr-14, in free and steroid-bound delta-5-3-ketosteroid isomerase were studied by determining the longitudinal and transverse 13C relaxation rates and nuclear Overhauser effects at both 500 and 600 MHz proton frequencies. Findings indicated that the amplitude of the high-frequency side chain movement of Tyr-14 was more restricted in the steroid complex compared to its backbone.

4-Oxalocrotonate tautomerase (4-OT) is an enzyme that catalyzes the isomerization of ketones, with the amino-terminal proline as the catalytic base. A study of the amino-terminal proline's role in isomerization by nuclear magnetic resonance spectroscopy using uniformly 15N-labeled 4-OT revealed that 3-bromopyruvate (3-BP) blocks the amino terminus of 4-OT to Edman degradation. Covalent bonding between a 15N resonance of 4-OT and the methylene carbon of the reduced adduct of 3-BP was also observed. Affinity labeling revealed the stoichiometry of a 4-OT monomer with 3-BP.

The pH dependence of the 4-oxalocrotonate tautomerase (4-OT)-catalyzed isomerization of ketones was examined by measuring the kinetics of 4-OT in different pH levels using 2-hydroxy-2,4-hexadiendioate and 2-hydroxy-2,4-pentadienoate as substrates. The pH dependences of the kinetic parameters for the 4-OT-catalyzed isomerization were bell-shaped with limiting sloped of unity on both sides of the pH optimum. An isomerization mechanism involving general base catalysis by the enzyme is discussed.

The tertiary structure of the Mut enzyme has been determined by distance geometry, simulated annealing and energy minimization with the program X-PLOR. The results indicate that the structure is globular and compact with the parallel portion of the beta-sheet sandwiched by two alpha-helices, forming an alpha + beta fold. A cluster of five glutamate residues form a region of electrostatic potential that constitute the metal binding site.

Nuclear magnetic resonance studies show that the binding of MutT enzyme to the nucleotides Mg2+ -AMPCPP and dGTP is anti conformation and this prevents intramolecular clashes when nucleotides with bulky substituents at the eighth position of the purine ring are hydrolyzed. The active site of MutT recognizes the nucleotides which prefer syn conformation during DNA transcription and MutT reduces the mutation rate in Escherichia coli by removing these nucleotides. It is possible that the anti conformation is essential for the MutT-catalyzed substitutions at the beta-phosphorous.

A heteronuclear NMR study of a denatured staphylococcal nuclease 131 residue fragment reveals that helix two and helix one retain characteristic helical structures, but also exhibit fast conformational changes between helical and extended conformations. The 83-86 and 94-97 segments exhibit properties associated with a native-like reverse-turn structure. The C-terminal alpha helix and the 5-strand beta-barrel indicate a hierarchical mechanism of nuclear folding.

A combination of 2D and 3D heteronuclear techniques were used to accomplish the assignment of the backbone NH, N, C-alpha, H-alpha, and CO resonances of the mutT enzyme. Ala C-alpha resonances were detected through a constant time 1H-13C HSQC experiment. The precise estimation of C-alpha chemical shifts in D2O, through HCACO and HCA(CO)N experiments and the precise matching of such changes with those in H2O through HNCA and HN(CO) CA studies, made the studies more convenient.

The backbone NMR assignments for H-alpha, C-alpha, HN, N and carbonyl C' resonances based on 2D and 3D homonuclear and heteronuclear NMR spectra and amide NH exchange data were used to establish the secondary structure of MutT in solution. A pair of alpha-helices, spanning residues 47-59 and 119-128, are evident in MuT. MuT's nine proline residues are trans.

Recombinant human tyrosine hydroxylase can be considered to be a metal-free apo enzyme (apo-hTHI) that is activated by binding with Fe 2+ and is competitively inhibited by binding with Co 2+ and Zn 2+, this binding influencing the enzyme's activity on phenylalanine. Studies of the paramagnetic effects of hTH1-Co2+, hTHI-Zn2+ and hTHI-Fe2+ on the protons of phenylalanine show that the enzyme augments the paramagnetic effects of Co2+ on the 1/T1 and 1/T2 values of phenylalanine protons, while LTHI-Zn2+ does not affect these values. The role of Fe2+ in the hydroxylation of phenylalanine is also studied.

Adenosine triphosphate is shown to be bound by PP-50, a peptide based on certain residues from the beta-subunit of mitochondrial F1-ATPase. PP-50 can bind ATP tightly at pH 4.0, when an NMR study of this phenomenon is possible.

A study presents the results of previous kinetic stereochemical, structural and binding studies which investigated the catalytic role of three arginine residues, namely, Arg-11, Arg-39 and Arg-61 in 4-oxalocrotonate tautomerase. Findings revealed that Arg-11 is important in both substrate binding and catalysis. Arg-39 contributes largely to catalysis while Arg-61 plays little or no role in substrate binding and catalysis. Experimental procedures and results are presented.