Alanine Variants Research Articles

Ca2+ Binding to the ExDxD Motif Regulates the DNA Cleavage Specificity of a Promiscuous Endonuclease

Most of the restriction endonucleases (REases) are dependent on Mg(2+) for DNA cleavage, and in general, Ca(2+) inhibits their activity. R.KpnI, an HNH active site containing ββα-Me finger nuclease, is an exception. In presence of Ca(2+), the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg(2+)-induced promiscuous activity. To elucidate the mechanism of unusual Ca(2+)-mediated activity, we generated alanine variants in the putative Ca(2+) binding motif, E(132)xD(134)xD(136), of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca(2+). We demonstrate that ExDxD residues are involved in Ca(2+) coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca(2+)-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca(2+) binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg(2+), the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca(2+) binding motif that confers site specific cleavage upon Ca(2+) binding is also critical for the promiscuous activity of the Mg(2+)-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

Exploring sequence requirements for C3/C4 carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β‐lactamase

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C₃/C₄ carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C₃/C₄ carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C₃/C₄ β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C₃/C₄ β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C₃/C₄ carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.

A natural peptide and its variants derived from the processing of infectious pancreatic necrosis virus (IPNV) displaying enhanced antimicrobial activity: A novel alternative for the control of bacterial diseases

The larger segment of the infectious pancreatic necrosis virus (IPNV) codifies most of the structural and non-structural proteins of the virus in two overlapping open reading frames (ORFs). The longer of the two ORF is expressed as a polyprotein which generates a number of variable length peptides of unknown function during processing. Since an appealing hypothesis would be that these peptides are generated by the virus to act as antimicrobial agents that favor viral infectivity in their fish host, we decided to test this possibility by selecting a master peptide and using it to generate substitution variants that may enhance their antimicrobial potential. A 20-residue master peptide (p20) was selected from the well-described maturation process of the structural viral protein VP2; several variants were then designed and chemically synthesized, ranging in size from 16 to 20 residues. The synthesized peptides were tested for in vitro activity against several prototype bacterial pathogens using standardized laboratory procedures. Chemically synthesized p20 and all its variants displayed broad activity against the tested bacteria and none of them were toxic to eukaryotic cells at least 10× the concentration used against the bacteria. Interestingly, when p20 was tested against the very aggressive bacterial pathogen Piscirickettsia salmonis, a common co-infectant of IPNV in salmonid fish, the specific activity of the novel peptide was significantly higher than that displayed for bactericidal fish farm antibiotics such as oxolinic acid, flumequine and florfenicol, which are commonly used to control Piscirickettsiosis in the field. It is potentially significant that the approach presented in this report provides a novel alternative for generating new and ideally more efficient and friendly safeguards for bacterial prophylaxis.

The Structures of the C185S and C185A Mutants of Sulfite Oxidase Reveal Rearrangement of the Active Site

Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 A resolution and to 2.4 A resolution in the presence of sulfite, and the C185A variant to 2.8 A resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier extended X-ray absorption fine structure spectroscopy (EXAFS) work on the human C207S mutant. An unexpected result was that in the C185S variant, in the absence of sulfite, the active site residue Tyr322 became disordered as did the loop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue relocalized to the active site. The C185A variant structure also indicated the presence of a third oxygen ligand; however, Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized Mo(VI) state in both the C185S and C185A variants of chicken SO and show the expected trioxodithiolene active site. Density functional theory calculations of the trioxo form of the cofactor reasonably reproducd the Mo horizontal lineO distances of the complex; however, the calculated Mo-S distances were slightly longer than either crystallographic or EXAFS measurements. Taken together, these results indicate that the active sites of the C185S and C185A variants are essentially catalytically inactive, the crystal structures of C185S and C185A variants contain a fully oxidized, trioxo form of the cofactor, and Tyr322 can undergo a conformational change that is relevant to the reaction mechanism. Additional DFT calculations demonstrated that such methods can reasonably reproduce the geometry and bond lengths of the active site.

Identification of the SSB binding site of Uracil DNA Glycosylase

Uracil DNA glycosylases (UDGs) catalyze the removal of uracil bases that arise in DNA by spontaneous cytosine deamination and dUTP misincorporation by DNA polymerases. UDGs are found in all kingdoms of life and in many cases have been shown to interact functionally with single‐strand DNA‐binding proteins (SSBs). The structural mechanisms that govern UDG/SSB interactions and the in vivo significance of the complex are currently unknown. We present the X‐ray crystal structures of Escherichia coli UDG in complex with the 9 C‐terminal residues of E. coli SSB and of Deinococcus radiodurans UDG in complex with the 12 C‐terminal residues of D. radiodurans SSB. The structures reveal a conserved platform on each UDG for binding SSB. Alanine variants of UDG residues involved in binding the SSB C‐terminal peptides lead to dramatic weakening of the cognate UDG/SSB complexes. These structures and solution studies provide firm evidence for the identification of the SSB interaction platform on UDG. Future genetic studies will examine the importance of UDG/SSB interactions in E. coli cells.

Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase

The c-type nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans is an integral membrane protein that catalyzes NO reduction; 2NO+2e(-)+2H(+)-->N(2)O+H(2)O. It is also capable of catalyzing the reduction of oxygen to water, albeit more slowly than NO reduction. cNORs are divergent members of the heme-copper oxidase superfamily (HCuOs) which reduce NO, do not pump protons, and the reaction they catalyse is non-electrogenic. All known cNORs have been shown to have five conserved glutamates (E) in the catalytic subunit, by P. denitrificans numbering, the E122, E125, E198, E202 and E267. The E122 and E125 are presumed to face the periplasm and the E198, E202 and E267 are located in the interior of the membrane, close to the catalytic site. We recently showed that the E122 and E125 define the entry point of the proton pathway leading from the periplasm into the active site [U. Flock, F.H. Thorndycroft, A.D. Matorin, D.J. Richardson, N.J. Watmough, P. Adelroth, J. Biol. Chem. 283 (2008) 3839-3845]. Here we present results from the reaction between fully reduced NOR and oxygen on the alanine variants of the E198, E202 and E267. The initial binding of O(2) to the active site was unaffected by these mutations. In contrast, proton uptake to the bound O(2) was significantly inhibited in both the E198A and E267A variants, whilst the E202A NOR behaved essentially as wildtype. We propose that the E198 and E267 are involved in terminating the proton pathway in the region close to the active site in NOR.

A Non-cleavable UmuD Variant That Acts as a UmuD′ Mimic

UmuD(2) cleaves and removes its N-terminal 24 amino acids to form UmuD'(2), which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the beta processivity clamp ("beta") of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif ((14)TFPLF(18)) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the beta-binding motif variant bind to beta with similar K(d) values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in beta is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic.

Kinetic characterization and ligand binding studies of His351 mutants of Bacillus anthracis adenylate cyclase

Edema factor is a calmodulin dependent adenylyl cyclase secreted as one of the primary exotoxins by Bacillus anthracis. A histidine residue at position 351 located in its active site has been implicated in catalysis but direct evidence of its functional role is still lacking. In the present study, we introduced mutations in full-length edema factor (EF) to generate alanine (H351A), asparagine (H351N), and phenylalanine (H351F) variants. Spectral analysis of these variants displayed no gross structural deformities. Kinetic characterization showed that the adenylyl cyclase activity of H351N and H351F mutants decreased 34- and 40-fold, respectively, whereas H351A mutant completely lost activity. K m and K i values for ATP, pH activity profiles, and calmodulin activation curves of asparagine and phenylalanine mutants were not altered markedly. This kinetic data corroborated our ligand binding studies. Apparent K d values for calmodulin and ATP binding were found to be similar for wild-type EF and these active site variants. The effective substitution of H351 by asparagine and phenylalanine, albeit at a greatly reduced K cat, without perturbing the ATP binding highlights the importance of this residue in transition-state stabilization. This was also evident from the positive free energy difference calculated for these mutants. However, equilibrium dialysis experiments revealed noticeable increase in ATP binding constant of H351A mutant, suggesting an additional role of H351 in precise substrate binding in the catalytic pocket. This is the first comprehensive study that describes the kinetic and ligand binding properties of H351 mutants and validates the importance of this residue in EF catalysis.

All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K+ Uptake by the KtrAB System from Vibrio alginolyticus

The subunit KtrB of bacterial Na+-dependent K+-translocating KtrAB systems belongs to a superfamily of K+ transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly70, Gly185, Gly290, and Gly402 from p-loops P(A) to P(D) of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB(A70), KtrB(A185), and KtrB(A290) maintained a substantial activity in KtrAB-mediated K+ uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on Vmax. Minor activities were also observed for three other variants: KtrB(A402), KtrB(S70), and KtrB(D185). With all of these variants, the property of Na+ dependence of K+ transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K+/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.

The Role of Histidines in the Acetate Kinase fromMethanosarcina thermophila

The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine --> alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.

Stabilization of the transition state for the transfer of tyrosine to tRNA Tyr by tyrosyl-tRNA synthetase

Aminoacylation of tRNA Tyr involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA Tyr. In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the α-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2′-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA Tyr substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA Tyr substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA Tyr, and suggest that catalysis of the second step of tRNA Tyr aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.

L, M and L–M hybrid cone photopigments in man: deriving λmax from flicker photometric spectral sensitivities

Using heterochromatic flicker photometry, we have measured the corneal spectral sensitivities of the X-chromosome-linked photopigments in 40 dichromats, 37 of whom have a single opsin gene in their tandem array. The photopigments encoded by their genes include: the alanine variant of the normal middle-wavelength sensitive photopigment, M(A180); the alanine and serine variants of the normal long-wavelength sensitive photopigment, L(A180) and L(S180); four different L–M hybrid or anomalous photopigments, L2M3(A180), L3M4(S180), L4M5(A180) and L4M5(S180); and two variants of the L-cone photopigment, encoded by genes with embedded M-cone exon two sequences, L(M2; A180) and L(M2; S180). The peak absorbances ( λ max) of the underlying photopigment spectra associated with each genotype were estimated by correcting the corneal spectral sensitivities back to the retinal level, after removing the effects of the macular and lens pigments and fitting a template of fixed shape to the dilute photopigment spectrum. Details of the genotype-phenotype correlations are summarized elsewhere (Sharpe, L. T., Stockman, A., Jägle, H., Knau, H., Klausen, G., Reitner, A. et al. (1998). J. Neuroscience, 18, 10053–10069). Here, we present the individual corneal spectral sensitivities for the first time as well as details and a comparison of three analyses used to estimate the λ max values, including one in which the lens and macular pigment densities of each observer were individually measured.

The interleukin-4 site-2 epitope determining binding of the common receptor gamma chain.

Human IL-4 (IL-4), one of the small four-helix-bundle cytokines, uses the specific IL-4 receptor a chain together with a promiscuous subunit, the common gamma chain (gamma(c)) for transmembrane signaling. The ligand-binding properties of gamma(c), which are presently poorly understood, were analysed by biosensor techniques employing recombinant ectodomains of gamma(c) and a receptor chains (IL4-BP). The formation of a ternary complex between solute gamma(c) ectodomain and IL-4 saturated IL4-BP could be established to exhibit a high dissociation constant Kd = 3 microM and a short half life tau1/2 = 7 s. This binding affinity resulted to the major part from the interaction of gamma(c) ectodomain with IL-4 and not from a direct contact of the ectodomains, since binding between solute gamma(c) ectodomain and IL-4 could be established (Kd about 150 microM), whereas no binding was found between the gamma(c) ectodomain and IL4-BP in the absence of IL-4. The IL-4 epitope involved in gamma(c) ectodomain interaction (site 2) was identified by means of an alanine-scanning mutational approach. The IL-4 site 2 comprised residues I11 and N15 on helix A together with Y124 on helix D as major binding determinants. The IL-4 alanine variants at site 2 generally showed only moderate defects in biological activity. Even the most affected variant [A124]IL-4 retained a partial agonist activity of about 50% during a T-cell proliferation assay. The dosis leading to a half-maximal response (EC50) was not altered by site-2 substitutions. The present results are in accordance with a two-step-dimerisation mechanism for IL-4 receptor activation, where solute IL-4 at physiological concentrations binds first via the high-affinity site 1 to the a chain only, since the affinity of IL-4 site 2 for gamma(c) is too low. This site-2 affinity seems to be sufficient, however, to promote, in a second step, a productive association of gamma(c) to an IL-4/alpha chain complex in the membrane.

A mixed-charge pair in human interleukin 4 dominates high-affinity interaction with the receptor alpha chain.

Human interleukin 4 (IL-4) binds to its cellular receptor with a Kd in the subnanomolar range, similar to many other 4-helix-bundle proteins interacting with members of the hematopoietin (cytokine) receptor superfamily. In the IL-4 system this interaction is predominantly determined by the extracellular domain (IL4-BP) of the receptor alpha chain (Kd approximately 150 pM). Now a high-resolution mutational and kinetic analysis has revealed that the high-affinity binding of IL-4 originates from a continuous patch of a few mostly polar or charged amino acid side chains located on helices A and C. The binding epitope comprises (i) a set of side chains determining the dissociation rate (k(off)) and (ii) a partially overlapping set determining the association rate constant (k(on)) of the IL-4/IL4-BP complex. The k(off) epitope is assembled from two juxtaposed main determinants (Glu-9 and Arg-88) surrounded by five side chains (Ile-5, Thr-13, Arg-53, Asn-89, and Trp-91) of lower importance. The cumulative increase in k(off) after alanine substitution is 10(5)-fold for the central mixed-charge pair and 3 x 10(3)-fold for the satellites. The k(on) epitope is formed by five positively charged residues on helix C (Lys-77, Arg-81, Lys-84, Arg-85, and Arg-88) and two neighboring residues on helix A (Glu-9 and Thr-13). The cumulative loss in k(on) of the alanine variants is only about 10-fold. These results provide the basis for an understanding of molecular recognition in cytokine receptor complexes and for an IL-4 antagonist design.

Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase

We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase. These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites. The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid). The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides. C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively. The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity. The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes). C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase. Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A. These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site.