Polymorphism p.Val231Ile alters substrate selectivity of drug-metabolizing arylamine N-acetyltransferase 2 (NAT2) isoenzyme of rhesus macaque and human

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes mediating the biotransformation of arylamine/arylhydrazine xenobiotics, including pharmaceuticals and environmental carcinogens. The NAT1 and NAT2 genes, and their many polymorphic variants, have been thoroughly studied in humans by pharmacogeneticists and cancer epidemiologists. However, little is known about the function of NAT homologues in other primate species, including disease models. Here, we perform a comparative functional investigation of the NAT2 homologues of the rhesus macaque and human. We further dissect the functional impact of a previously described rhesus NAT2 gene polymorphism, causing substitution of valine by isoleucine at amino acid position 231. Gene constructs of rhesus and human NAT2, bearing or lacking non-synonymous polymorphism c.691G>A (p.Val231Ile), were expressed in Escherichia coli for comparative enzymatic analysis against various NAT1- and NAT2-selective substrates. The results suggest that the p.Val231Ile polymorphism does not compromise the stability or overall enzymatic activity of NAT2. However, substitution of Val231 by the bulkier isoleucine appears to alter enzyme substrate selectivity by decreasing the affinity towards NAT2 substrates and increasing the affinity towards NAT1 substrates. The experimental observations are supported by in silico modelling localizing polymorphic residue 231 close to amino acid loop 125–129, which forms part of the substrate binding pocket wall and determines the substrate binding preferences of the NAT isoenzymes. The p.Val231Ile polymorphism is the first natural polymorphism demonstrated to affect NAT substrate selectivity via this particular mechanism. The study is also the first to thoroughly characterize the properties of a polymorphic NAT isoenzyme in a non-human primate model.     

Free energy analysis of ω-transaminase reactions to dissect how the enzyme controls the substrate selectivity

ω-Transaminase (ω-TA) is the only naturally occurring enzyme allowing asymmetric amination of ketones for production of chiral amines. The active site of the enzyme was proposed to consist of two differently sized substrate binding pockets and the stringent steric constraint in the small pocket has presented a significant challenge to production of structurally diverse chiral amines. To provide a mechanistic understanding of how the (S)-specific ω-TA from Paracoccus denitrificans achieves the steric constraint in the small pocket, we developed a free energy analysis enabling quantification of individual contributions of binding and catalytic steps to changes in the total activation energy caused by structural differences in the substrate moiety that is to be accommodated by the small pocket. The analysis exploited kinetic and thermodynamic investigations using structurally similar substrates and the structural differences among substrates were regarded as probes to assess how much relative destabilizations of the reaction intermediates, i.e. the Michaelis complex and the transition state, were induced by the slight change of the substrate moiety inside the small pocket. We found that ≈80% of changes in the total activation energy resulted from changes in the enzyme-substrate binding energy, indicating that substrate selectivity in the small pocket is controlled predominantly by the binding step (K_M) rather than the catalytic step (k_cat). In addition, we examined the pH dependence of the kinetic parameters and the pH profiles of the K_M and k_cat values suggested that key active site residues involved in the binding and catalytic steps are decoupled. Taken together, these findings suggest that the active site residues forming the small pocket are mainly engaged in the binding step but not significantly involved in the catalytic step, which may provide insights into how to design a rational strategy for engineering of the small pocket to relieve the steric constraint toward bulky substituents.     

Engineering Neprilysin Activity and Specificity to Create a Novel Therapeutic for Alzheimer’s Disease

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer’s disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer’s disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1–40, 1–42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1–40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer’s disease.     

Disparity in productive binding mode of the slow-reacting enantiomer determines the novel catalytic behavior of Candida antarctica lipase B

In the context of specifying the origin of enzyme enantioselectivity, the present study explores the lipase enantioselectivity towards secondary alcohols of similar structure from the perspective of substrate binding. By carrying out molecular mechanics minimization as well as molecular dynamics simulation on tetrahedral reaction intermediates which are used as a model of transition state, we identify an unconventional productive binding mode (PBM)—M/H permutation type for Candida antarctica lipase B (CALB). The in silico results also indicate that different PBMs of the slow-reacting enantiomer do exist in one lipase even when there is little structural differences between substrates, e.g. compounds with Ph or CH₂CH₂Ph group display the M/H permutation type PBM while molecules with CH₂Ph show the M/L permutation type PBM. By relating the PBMs of substrates to the experimentally determined E-values obtained by Hoff et al. [16], we find that disparity in PBM of the slow-reacting enantiomer determines why E-values of substrates with CH₂Ph were lower than E-values of substrates with Ph or CH₂CH₂Ph group. The modeling results also suggest that the “pushed aside” effect of the F atom and Br atom accommodates the medium size substituent of the substrate better in the stereospecificity pocket of the enzyme.     

Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome

From a biogas reactor metagenome an ORF (bp_cel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BP_Cel9A exhibited?≤?55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BP_Cel9A hydrolyzed the beta-1,3–1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (>?1%). In contrast to BP_Cel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BP_Cel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg²⁺ may adopt the function of Ca²⁺. BP_Cel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.     

A double mutant of highly purified Geobacillus stearothermophilus lactate dehydrogenase recognises l-mandelic acid as a substrate

Lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) (bsLDH) has a crucial role in producing chirally pure hydroxyl compounds. α-Hydroxy acids are used in many industrial situations, ranging from pharmaceutical to cosmetic dermatology products. One drawback of this enzyme is its limited substrate specificity. For instance, l-lactate dehydrogenase exhibits no detectable activity towards the large side chain of 2-hydroxy acid l-mandelic acid, an α-hydroxy acid with anti-bacterial activity. Despite many attempts to engineer bsLDH to accept α-hydroxy acid substrates, there have been no attempts to introduce the industrially important l-mandelic acid to bsLDH. Herein, we describe attempts to change the reactivity of bsLDH towards l-mandelic acid. Using the Insight II molecular modelling programme (except ‘program’ in computers) and protein engineering techniques, we have successfully introduced substantial mandelate dehydrogenase activity to the enzyme. Energy minimisation modelling studies suggested that two mutations, T246G and I240A, would allow the enzyme to utilise l-mandelic acid as a substrate. Genes encoding for the wild-type and mutant enzymes were constructed, and the resulting bsLDH proteins were overexpressed in Escherichia coli and purified using the TAGZyme system. Enzyme assays showed that insertion of this double mutation into highly purified bsLDH switched the substrate specificity from lactate to l-mandelic acid.     

Glutathione transferases in primary rat hepatomas: the isolation of a form with GSH peroxidase activity

A previously uncharacterized glutathione (GSH) transferase which is not apparent in normal liver, accounts for at least 25% of the soluble GSH transferase content of primary hepatomas induced by feeding N,N-dimethyl-4-aminoazobenzene. This enzyme is readily isolated, has an isoelectric point of 6.8, is composed of two identical subunits of apparent M_r 26 000 and has GSH transferase activity towards a number of substrates including benzo(a)pyrene-7,8-diol-9,10-oxide. It is unusual in that it has GSH peroxidase activity towards fatty acid hydroperoxides but not towards the model substrates, cumene hydroperoxide and t-butyl hydroperoxide. It has been shown by tryptic peptide analysis to be distinct from GSH transferases composed of subunits 1, 2, 3,4 or 6 and has been designated GSH transferase 7-7.     

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.     

The isolation of a fetal rat liver glutathione S-tranferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Y_c (M_r 28 000) and a subunit (M_r 25 500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit ‘Y_fetus’. The enzyme which we term glutathione S-transferase Y_cY_fetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

Vanadium containing bromoperoxidase - Insights into the enzymatic mechanism using X-ray crystallography

The X-ray crystal structure of the vanadium bromoperoxidase from the red algae Corallina pilulifera has been solved in the presence of the known substrates, phenol red and phloroglucinol. A putative substrate binding site has been observed in the active site channel of the enzyme. In addition bromide has been soaked into the crystals and it has been shown to bind unambiguously within the enzyme active site by using the technique of single anomalous dispersion. A specific leucine amino acid is seen to move towards the bromide ion in the wild-type enzyme to produce a hydrophobic environment within the active site. A mutant of the enzyme where arginine 397 has been changed to tryptophan, shows a different behaviour on bromide binding. These results have increased our understanding of the mechanism of the vanadium bromoperoxidases and have demonstrated that the substrate and bromide are specifically bound to the enzyme active site.     

Isolation and divalent-metal activation of a β-xylosidase,RUM630-BX

The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (k_cat) by divalent metals Mg²⁺, Mn²⁺ and Co²⁺ but not by Ca²⁺, Ni²⁺, and Zn²⁺. Activation of RUM630-BX by Mg²⁺ (t_0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (k_cat/K_m) of 299 s⁻¹ mM⁻¹ on xylotetraose being the highest reported.     

A chemoenzymatic route to synthesize unnatural sugar nucleotides using a novel N-acetylglucosamine-1-phosphate pyrophosphorylase from Camphylobacter jejuni NCTC 11168

A novel N-acetylglucosamine-1-phosphate pyrophosphorylase was identified from Campylobacter jejuni NCTC 11168. An unprecedented degree of substrate promiscuity has been revealed by systematic studies on its substrate specificities towards sugar-1-P and NTP. The yields of the synthetic reaction of seven kinds of sugar nucleotides catalyzed by the enzyme were up to 60%. In addition, the yields of the other nine were around 20%. With this enzyme, three novel sugar nucleotide analogs were synthesized on a preparative scale and well characterized.     

Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes

Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/φ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/φ)(T/S)(D/E)pY(G/A/S/Q) (where φ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X_1–6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp¹⁶⁴, which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo.     

Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg⁻¹ and nearly 8-folds higher k_cat/K_m value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.     

Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO₂/O₂ specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.     

How is the reactivity of laccase affected by single-point mutations? Engineering laccase for improved activity towards sterically demanding substrates

In spite of its broad specificity among phenols, Trametes versicolor laccase hardly succeeds in oxidizing hindered substrates. To improve the oxidation ability of this laccase towards bulky phenolic substrates, we designed a series of single-point mutants on the basis of the amino-acid layout inside the reducing substrate active site known from the crystal structure of the enzyme. Site-directed mutagenesis has addressed four phenylalanine residues in key positions 162, 265, 332, and 337 at the entrance of the binding pocket, as these residues appeared instrumental for docking of the substrate. These phenylalanines were replaced by smaller-sized but still apolar alanines. A double mutant F162A/F332A was also designed. Measurement of the oxidation efficiency towards encumbered phenols has shown that mutant F162A was more efficient than the wild-type laccase. The double mutant F162A/F332A led to 98% consumption of bisphenol A in only 5 h and was more efficient than the single mutants in the aerobic oxidation of this bulky substrate. In contrast, lack of appropriate hydrophobic interactions with the substrate possibly depresses the oxidation outcome with mutants F265A and F332A. One explanation for the lack of reactivity of mutant F337A, supported by literature reports, is that this residue is part of the second coordination shell of T1 Cu. A mutation at this position thus leads to a drastic coordination shell destabilization. Thermal stability of the mutants and their resistance in a mixed water–dioxane solvent have also been investigated.     

Cloning,expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida

l-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid substrates when combined with abiotic reductants. The gene nadB encoding the l-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K_M for l-aspartic acid of 2.26 mM and a k_cat = 10.6 s⁻¹, with lower activity also displayed towards l-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 °C, but rapid losses in activity were observed at 50 °C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both l-asparagine and l-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.     

The heterogeneity of uridine diphosphate glucuronyltransferase from rat liver

1. The glucuronide conjugation of p-nitrophenol, phenolphthalein, o-aminophenol and 4-methylumbelliferone by rat liver microsomes has been studied. The detergent Triton X-100 activated UDP-glucuronyltransferase activity towards all these substrates, therefore the optimum activating concentration was added in all experiments. 2. Mg²⁺ enhanced the conjugation of the substrates. 3. With phenolphthalein substrate inhibition occurred but this could be relieved by adding albumin, which binds excess of phenolphthalein. 4. Kinetic constants of the substrates and UDP-glucuronate have been determined. Mutual inhibition was found with the substrates p-nitrophenol, 4-methylumbelliferone and phenolphthalein. p-Nitrophenol conjugation was inhibited competitively by phenolphthalein and 4-methylumbelliferone. 5. o-Aminophenol did not inhibit the conjugation of the other three substrates because these are conjugated preferentially to o-aminophenol. 6. It is concluded that the four substrates are conjugated by one enzyme at the same active site.     

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp²³⁰ residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.     

Substrate-mediated increased reactivity of a critical sulfhydryl group of crystals of cytoplasmic aspartate transaminase

The extramitochondrial isozyme of aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1) contains a cysteinyl residue (cysteine-390) which, in the presence of substrate, displays enhanced reactivity toward sulfhydryl reagents. To gain insight into the structural similarity of the enzyme in solution compared to its crystalline state and into the type of structural change induced by substrates, the reactivity of Cys-390 in the crystalline enzyme has been studied. The flat yellow plates, crystallized from polyethylene glycol, form spectroscopically detectable enzyme-substrate complexes (C. M. Metzler, D. E. Metzler, D. S. Martin, R. Newman, A. Arnone, and P. Rodgers, 1978, J. Biol. Chem. 253, 5251–5254). The crystals, both in the presence and absence of the substrate pair, glutamate and α-ketoglutarate, were treated with N-ethylmaleimide or N-ethyl[1-¹⁴C]maleimide and the extent of the reaction was monitored by the colorimetric sulfhydryl reaction with 5,5′-dithiobis-2-nitrobenzoic acid, by amino acid analysis, by radioactivity incorporated, and by the measurement of enzyme activity. A cysteine residue was modified only in the presence of substrate; the crystals remained undamaged. Since, any large conformational change in the enzyme would either be prevented by the crystalline lattice or would disrupt its integrity, it is concluded that the enhanced reactivity of cysteine-390 in the presence of substrates must be due to only a small local conformational change in the substrate binding region.