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The structural determinants of substrate specificity of human cytochrome P450 2C8(CYP2C8) are investigated using site-directed mutants chosen based on a preliminary substrate pharmacophore and a three-dimensional (3D) model.

3 alpha-Hydroxysteroid dehydrogenases (3 alpha-HSDs) catalyze the interconversion between 5 alpha-dihydrotestosterone (5 alpha-DHT), the most potent androgen, and 3 alpha-androstanediol (3 alpha-diol), a weak androgen metabolite. It is found that discrete point mutations in the cofactor and steroid binding site can alter the rate-determining step for the NADP(super +)-dependent oxidation of 3 alpha-diol.

Point mutational study reveals that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit contribure a 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Further, residues Lys-34, Gly-57, Asp-59 and Asp-173 contribute to the high affinity of the alpha-epsilon site than that of alpha-delta site.

In Drosophila comatose, the sec 18-1 mutation, in terms of G89D substitution in the Sec 18p domain, affects the protein's ability to bind to Sec17p of the yeast soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein. Data suggest that NSF protein binds to soluble NSF attachment protein and it is dependent on ATPase stimulation.

The mutational analysis of the presumptive nucleolytic center of McrC subunit of the McrBC system of Escherichia coli suggests that McrC mediates DNA cleavage by McrBC. The catalytic center of the McrC is represented by the sequence TD(sup)224 ....D(sup)257 AK.

Results suggest that the 'pol beta-like' region of p58 subunit of human DNA primase participates in the initiation of primer synthesis, its elongation and translocation, and counting. Mutations in the p58 region affects both initiation and elongation of primers.

Results show that mutants Glu820 and Glu795 of the rat gastric proton, potassium- ATPase exhibit ATPase activity constitutively and increased ATP phosphorylation as shown by double mutations, involving replacement of glutamate residues with glutamine and alanine at positions 795 and 820, respectively.

Results show that the activity restoration in the mutant thymidylate synthase follows a sequential pattern of subunit separation and their subsequent recombination to an active heterodimer. Sequence data of the homo- and heterodimers of the enzyme indicate that the active enzyme is a product of subunit exchange.

Research demonstrates that annexin-IV mediated membrane aggregation occurs in vivo through phosphorylation and dephosphorylation as shown by site-directed mutagenesis and mutant annexin-IV analysis. Data indicate that phosphorylation influences annexin-IV structure, self-association, and inter-liposome junction formation during membrane aggregation.

Research has been conducted on the binding of the component B to the hydroxylase component. The identification of the amino acid residues of the component B that affect the interconversion of the methane monooxygenase cycle intermediates has been conducted and results are presented.

Research has been conducted on the regulatory cAMP-dependent protein kinase R-subunit. The role of the tryptophan and the adjacent domain in domain A stabilization and folding is discussed.

Research has been conducted on the nucleotide-induced conformational changes in wild-type P-glycoprotein. The monitoring of these changes by the trypsin digestion has been carried out.

Research has been conducted on the protein stability in vitro. The effect of glycerol addition on the A (sub)2 folding thermodynamics and cavity mutants has been investigated.

Research has been conducted on the Rhodobacter capsulatus cytochrome c (sub)2 Gly 34 and Pro 35. The characterization of Gly 34 mutation to Ser in Rb. capsulatus cytochrome c(sub)2 is described.

Research has been conducted on the glucose transporter IICB (super)Glc subunit. The mechanism that couples vectorial translocation with substrate phosphorylation is described.

Research has been conducted on the R-plasmid-encoded enzyme R67 dihydrofolate reductase (DHFR). Results indicate that this enzyme has no structural homology to the chromosomal DHFRs.

Research has been conducted on the hyperthermophilic archaeon Pyrococcus furiosus beta-glucosidase. A library of random CelB mutants in Escherichia coli has been generated.

Research has been conducted on Salmonella typhimurium phosphoenolpyruvate enyme mutants. The identification of these mutants as Arg126 has been carried out.

Research has been conducted on the protein folding reaction. The changes in CD2.d1 folding reaction heat capacity, entropy and enthalpy have been measured.

Research has been conducted on the CC chemokine structures. The use of nuclear magnetic resonance spectroscopy in analyzing the mutants' oligomeric state is described.

Research has been conducted on the single Trp mutants of class B Tet repressor. The engineering of these mutants to investigate loop dynamics has been carried out.

Research has been conducted on the human interferon-beta-1a (IFN-beta). The identification of the molecule regions important for receptor binding and for functional activity has been performed.

Research has been conducted on the 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction. The results of experiments with Lactobacillus casei enzyme tryptophan 82 mutants are presented.

Research has been conducted on the proximal heme iron ligands electronic characteristics in heme proteins. The role of the ligands in oxygen and peroxide activation has been investigated.

Research has been conducted on the glutamic acid Glu (super)795. The generation and expression of mutants in Sf9 insect cells are described.

Research has been conducted on the mechanism of the muscle contraction trough tropomyosin and troponin activated by Ca (super)2+. The use of Dictyostelium discoideum in producing mutant actins is described.

Research has been conducted on the effect of histidine ligand of human transferin on iron binding. Site-directed mutagenesis, spectroscopy and structural analysis are examined.

A theoretical and experimental study was performed to determine influence of the deuterium kinetic isotope and the mechanism behind a bacterial luciferase reaction. The isotope effect was found to be insignificantly influenced by the use of alphaC106A and alphaC106S site-directed mutants, chlorine replacement at the 8-location of the reduced flavin, changes in the hydrogen-ion concentration or changes in the length of the aldehyde chain. The findings suggested that the mechanism of rate-limiting electron transfer corresponds to the enigmatic aldehydic isotope effect.

Motional freedom in the native (N) state and rotational freedom in the unfolded state characterize the motional dynamics of a single tryptophan (W53) buried in a mutant barstar. Time-resolved fluorescence shows that W53 has one lifetime in the N-state but three lifetimes in a molten globule-like low-pH (A) form. Fluorescence quenching results in solvent W53 being inaccessible in the N-state and accessible in the A-form. Denaturation analysis shows that fluorescence anisotropy is more useful than both circular dichroism and fluorescence intensity in detecting the unfolding transitions.

Mutations in the B(sub 12)-binding region of methionine synthase decrease the reactivity of methylcobalamin cofactor. Visible absorbance spectroscopic and electron paramagnetic spectroscopic analyses indicate that mutation at histidine 759 inhibits the turnover rate and reduces the coordination strength of the upper oxygen ligand. Mutations in histidine 759, aspartate 757, and serine 810 increase the rate of AdoMet-mediated reactivation of cob(II)alamin enzyme. Nitrogen ligand in lower axial location is necessary for heterolytic methyl transfer.

A synthetic gene module constructed in metH gene analyzes the role of amino acid residues in the cobalamin-binding region of Escherichia coli methionine synthase. The module contains specific restriction sites at nearly 80 base pairs. The expression of methionine synthase is independent of the introduction of synthetic module. Seven mutant proteins are constructed from this synthetic module using cassette mutagenesis. The His759Gly mutation inhibits steady-state activity of the cobalamin. The characteristic influences of seven mutant proteins are discussed.

Mutagenesis within an M13 phage vector alters the specificity and activity of the Enterobacter cloacae P99 beta-lactamase. Mutations among positions 286-290 affect enzyme specificity for the 3'-substituted cephalosporins and stabilize the enzyme. Amino acids in the 286-290 region of E. cloacae enzyme play no part in the catalytic activity, though they are close to the active site of the enzyme. Michaelis-Menten kinetic analysis with cephalosporin indicates that amino acids in the region are susceptible to substitution.

Substitution of histidine 269 with ligands with a divalent metal ion, n2, in the enzyme, glutamine synthetase from Escherichia coli has little effect on ligand-binding. Glutamine synthetase from Escherichia coli catalyzes the conversion of glutamate to glutamine in the presence of ATP and ammonia, and requires two divalent metal ions, n1 and n2. The negative charge around the n2 metal ion due to glutamate 269 decreases the ability of the n2 metal ion to allow glutamate binding in the presence of ATP. The charge lowers the interactions between metal ion and reactants in the transition state.

The binding of the prodomain to native subtilisin accelerates folding, indicating that the subtilisin intermediate has structural characteristics of the native form. The catalytic activity is unaffected by binding of native subtilisin. The binding site of the prodomain is different from its site on the native protein. An analysis of mutations in the C-terminal region of the prodomain reveals that the binding site is in 100-144 amino acid sequence of subtilisin. The amino acids probably have a native-like fold in the intermediate which is stabilized by the prodomain.

The lysine residues at positions 296 and 206 in the N-lobe of recombinant human serum transferrin (hTF/2N) are necessary for the binding of divalent anions. A study conducted on the binding of synergistic anions to the wild-type and mutant protein using ultraviolet spectroscopy indicates that the binding is proportional to pH. The binding of anions to hTF/2N occurs with the help of a proton. The mutation has little effect on the binding of synergistic anions such as oxalate and malonate and no effect on the binding of nonsynergistic anions such as phosphate and sulfate.

A study of the structural basis for enhancing catalytic activity of mutant E165D chicken triosephosphate isomerase by secondary mutation, S96P, reveals that alteration in enzyme active site initiated by site directed mutation may change the location of attached water molecules. S96P mutation is responsible for the loss of two water molecules at 165 position in the isomerase which results in an increase in hydrophobicity and the pKa rate. A combination of an increase in basicity of the enzyme and acidity of the proton substrate increases the activity of the isomerase.

Thr-119-Met mutation stabilizes the tetrameric transthyretin (TTR) toward acid denaturation and thereby induces reduced amyloidogenicity. Val-30-Met, Val-30-Met/Thr-119-Met and Thr-119-Met tetrameric variants of TTR are expressed using Escherichia coli system. Under moderate acidic denaturing conditions and low concentrations, the FAP variants assemble into amyloid fibrils. The denaturation curve for Thr-119-Met variant mediated by fluorescence revealed no substantial concentration of amyloidogenic intermediate indicating that the intermediate is unstable.

Pseudomonas aeruginosa strains PAK and KB7 have differences between their adhesintopes, indicating the existence of compensatory mutations among pilin strains. Structural analysis of the pilins' adherence-binding domains showed this. The interactions between the pilin ligand and the A549 cell surface receptors depends on the conformation mediated by the disulphide bridges. The structural aspects of pilin-receptor interactions are discussed.

Analysis of 21 consecutive residues in the M1 segment of the mouse ACh receptor alpha subunit reveals that M1 is exposed in the channel and this exposure is altered on adding ACh. Cysteine is available to positively charged, sulfihydryl-specific reagents in 10 out of 21 mutated positions. The N-terminal of alpha-M1 mediates the lining of the channel indicating that the structure of M1 is random. The experimental procedure of the analysis is discussed.

Site-directed mutagenesis of Escherichia coli fumarate reductase in which FrdB Cys148, Cys151, Cys154 and Cys158 are substituted by Ser reveals that the three clusters are present in FrdB subunit with Cys57, Cys65, Cys62, Cys77 ligating the (2Fe-2S) cluster, Cys148, Cys151 and Cys214 ligating the (4Fe-4S) cluster and Cys158, Cys204, and Cys210 ligating (3Fe-4S) cluster. Enzymes with very low activity and dissociation are produced from the cytoplasmic membrane due to these mutations. EPR analysis indicates that the three Fe-S clusters are aggregated and spectroscopic characteristics are unchanged.

Mutation of Gly90, Glu113, Ala292, and Lys296 in the rhodopsin pigment constitutively activates G protein transducin, but the mutants are constitutively phosphorylated by rhodopsin kinase. The phosphorylated mutants bind arrestin in a reaction that prevents the interaction with transducin. Thus the same mutations which activate the transducin also cause phosphorylation by rhodopsin kinase and the inhibition by arrestin binding.

Second-site suppressor mutations for the Asp-66 convergent Cys mutant of the transposon Tn10-encoded metal-tetracyline/H+ antiporter of Escherichia coli demonstrates that a charge-neutralizing pair is formed. Asp 66 is the only important acidic residue of the transposon Tn10-encoded metal-tetracyline/H+ antiporter. The nature of the second-site suppression for Cys66 mutant is different from the nature of the suppressions for the Asn285 mutant with Glu220 mutation.

Mutation of cysteines that constitutes the active site disulfide in lipoamide dehydrogenase to a serine residue to give the altered enzymes, C44S and C49S, helps to study the redox behavior of the flavin adenine dinucleotide (FAD) in the absence of the disulfide. The redox potential of the FAD in C44S and C49S is -379 and -345 mV, respectively. The redox potential of the FAD in C49S, C44S, and in wild type enzyme is moderated by the electronegativity of its nearest neighbor, hydroxyl, thiolate, or disulfide.

A study analyzes the role of arginines 54 and 305, aspartic acid 309, and tryptophan 317 located at subsites 1 and 2 in glucoamylase in catalysis and substrate recognition. Arg305 is a key determinant in glucoamylase (GA) for the recognition of alpha-1,4-linked substrates. In case of Trp317 which stacks with Arg305, any minute structural change effects both the substrate recognition ability and the protein stability. These mutants have reduced stability at elevated temperature and in guanidine hydrochloride.

The enzymatic activity of ribonuclease HI (RNase HI) is not much affected by the simultaneous introduction of five mutations and the thermostability of the enzyme increases. Crystal structure studies of the mutant proteins reveal that the local conformational change is responsible for the stability of the enzyme. The mutant enzyme shows greater resistance to acid denaturation and proteolysis, which might increase the applications of mutant RNase HI.

The helical stacks (HS) of recombinant (r)-protein C (PC) and r-activated protein C (APC) are needed for the proper alignment of the active site of the enzyme with the cleavage site of the substrate. Human PC is the precursor of the serine protease APC and is activated by human coagulation factor II and inhibited by calcium ions. APC is an anticoagulant which can cleave peptide bonds. Mutations prepared with substitutions in the amino acids in the HS of PC indicate that the HS is more important than the amino acid sequence in the calcium ion dependent reactions.

A short hydrogen bond exist between Glu-104 and the inhibitor during the inhibitory complex formation between Escherichia coli cytidine deaminase and the transition-state analog 3,4-dihydrouridine. The activation barrier for deamination is reduced because of the hydrogen bonding in carboxymethyl group of Glu-104 which stabilizes the altered substrate in the transition state. The enzyme-substrate and enzyme-product complexes are destabilized. The low catalytic activity of the mutant enzyme is elevated in the presence of formate ion.

Mutational analysis of the beta-72 site of Agmenellum quadruplicatum C-phycocyanin to establish the role of the wild-type N5-methylasparagine present at the beta-72 site suggests that N5-methylasparagine is required for the environment around the beta-84 fluorescing bilin tetrapyrrole chromophore. The new environment will restrict the rates of nonradiative energy losses that have the potential to abolish the high quantum yield for energy transfer.

Kinetic studies of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes help analyze the generality of the NUX rule for the GUC triplet near the cleavage site in the ribozymes. The results suggest that triplets such as GUC, GUA and AUA, associated with cis reactions, possess high k(sub cat) values, while only AUA and GUA possess high K(sub m) values.

PCR cassette mutagenesis helps design zinc-finger yeast ADR1 mutants with different binding specificity to 16 UAS1 variants of thymine bases T5 and T6. Substitution of His for Leu146 decreases the binding of wild type UASI having T6, but decreases the binding to sequences having purine A6 or G6. Substitution of Lys for Leu146 increases the binding of both G6 and T6. Analysis of mutants with Glu for Arg 149 confirms the interaction of Arg 149 with G5 or T5 in UAS1 DNA.