Characterization of a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 by biochemical, X-ray crystallographic, and mass spectrometric approaches.

We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.     

Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1): NQO1 inhibition and growth inhibitory activity in human pancreatic MIA PaCa-2 cancer cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) is currently an emerging target in pancreatic cancer. In this report, we describe a series of indolequinones, based on 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), and evaluate NQO1 inhibition and growth inhibitory activity in the human pancreatic MIA PaCa-2 tumor cell line. The indolequinones with 4-nitrophenoxy, 4-pyridinyloxy, and acetoxy substituents at the (indol-3-yl)methyl position were NADH-dependent inhibitors of recombinant human NQO1, indicative of mechanism-based inhibition. However, those with hydroxy and phenoxy substituents were poor inhibitors of NQO1 enzyme activity, due to attenuated elimination of the leaving group. The ability of this series of indolequinones to inhibit recombinant human NQO1 correlated with NQO1 inhibition in MIA PaCa-2 cells. The examination of indolequinone interactions in complex with NQO1 from computational-based molecular docking simulations supported the observed biochemical data with respect to NQO1 inhibition. The design of both NQO1-inhibitory and noninhibitory indolequinone analogues allowed us to test the hypothesis that NQO1 inhibition was required for growth inhibitory activity in MIA PaCa-2 cells. ES936 and its 6-methoxy analogue were potent inhibitors of NQO1 activity and cell proliferation; however, the 4-pyridinyloxy and acetoxy compounds were also potent inhibitors of NQO1 activity but relatively poor inhibitors of cell proliferation. In addition, the phenoxy compounds, which were not inhibitors of NQO1 enzymatic activity, demonstrated potent growth inhibition. These data demonstrate that NQO1 inhibitory activity can be dissociated from growth inhibitory activity and suggest additional or alternative targets to NQO1 that are responsible for the growth inhibitory activity of this series of indolequinones in human pancreatic cancer.     

Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols

The mechanism-based inhibition of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding [DeWolf, W. E., Jr., & Kruse, L. I. (1985) Biochemistry 24, 3379]. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3096] p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate "activator" addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.     

Triazoloacridin-6-ones as novel inhibitors of the quinone oxidoreductases NQO1 and NQO2

A range of triazoloacridin-6-ones functionalized at C5 and C8 have been synthesized and evaluated for ability to inhibit NQO1 and NQO2. The compounds were computationally docked into the active site of NQO1 and NQO2, and calculated binding affinities were compared with IC₅₀ values for enzyme inhibition. Excellent correlation coefficients were demonstrated suggesting a predictive QSAR model for this series of structurally similar analogues. From this we have identified some of these triazoloacridin-6-ones to be the most potent NQO2 inhibitors so far reported.     

Suicide-inhibitory bifunctionally linked substrates (SIBLINKS) as phospholipase A2 inhibitors. Mechanistic implications

Novel phospholipids that function as mechanism-based inhibitors for phospholipase A2 (PLA2) are described. PLA2-catalyzed hydrolysis of the sn-2 ester of these suicide-inhibitory bifunctionally linked substrates (SIBLINKS) followed by a cyclization reaction generates a cyclic anhydride at the active site of the enzyme which leads to inhibition. Structure/activity relationships for the SIBLINKS substituents in the sn-1 and sn-2 position are delineated. Time courses and efficiency of SIBLINKS inhibition are reported and compared for extracellular PLA2s obtained from Naja naja naja, porcine pancreas, bee venom, Crotalus atrox and Crotalus adamanteus. SIBLINKS-inhibited PLA2s cannot process either monomeric or micellar substrates consistent with inhibition at the catalytic site. Some SIBLINKS efficiently inactivate 1 mol of N. naja naja and C. adamanteus PLA2/6-10 mol of SIBLINKS hydrolyzed. Inhibition of N. naja naja PLA2 can be reversed by hydroxylamine, suggesting that a tyrosine residue is acylated.     

Flavin adenine dinucleotide content of quinone reductase 2: analysis and optimization for structure-function studies

Quinone reductase 2 (NQO2) is a broadly expressed enzyme implicated in responses to a number of compounds, including protein kinase inhibitors, resveratrol, and antimalarial drugs. NQO2 includes a flavin adenine dinucleotide (FAD) cofactor, but X-ray crystallographic analysis of human NQO2 expressed in Escherichia coli showed that electron density for the isoalloxazine ring of FAD was weak and there was no electron density for the adenine mononucleotide moiety. Reversed-phase high-performance liquid chromatography (HPLC) of the NQO2 preparation indicated that FAD was not present and only 38% of the protomers contained flavin mononucleotide (FMN), explaining the weak electron density for FAD in the crystallographic analysis. A method for purifying NQO2 and reconstituting with FAD such that the final content approaches 100% occupancy with FAD is presented here. The enzyme prepared in this manner has a high specific activity, and there is strong electron density for the FAD cofactor in the crystal structure. Analysis of NQO2 crystal structures present in the Protein Data Bank indicates that many may have sub-stoichiometric cofactor content and/or contain FMN rather than FAD. This method of purification and reconstitution will help to optimize structural and functional studies of NQO2 and possibly other flavoproteins.     

Factors, determining the effectiveness of tryptophanase interaction with amino acids

Inhibition of tryptophanase-catalyzed decomposition of S-(o-nitrophenyl)-L-cysteine by a variety of amino acids has been investigated. For amino acids similar to the natural substrate and for those having minimal steric requirements for the side chain, the linear correlation exists between-RTlnKi and side chain hydrophobicity. L-ornithine and L-arginine are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This can be explained by an interaction between a positively charged group of the side chain of L-arginine or L-ornithine and a nucleophilic group of the active site. The comparison of affinity of tryptophanase for L-phenylalanine and L-homophenylalanine indicates that there is a special locus in the active site where aromatic groups are bound and oriented approximately parallel to the cofactor plane experiencing no steric hindrance. For a large number of amino acids the rates of the enzymic alpha-proton exchange in 2H2O are comparable with the rate of the reaction with L-tryptophan. Very low rate of alpha-proton exchange observed with L-alanine is an exception.     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone

Background

Multimeric naphthoquinones are redox-active compounds that exhibit antineoplastic, antiprotozoal, and antiviral activities. Due to their multimodal effect on perturbation of cellular oxidative state, these compounds hold great potential as therapeutic agents against highly proliferative neoplastic cells. In our previous work, we developed a series of novel dimeric naphthoquinones and showed that they were selectively cytotoxic to human acute myeloid leukemia (AML), breast and prostate cancer cell lines. We subsequently identified the oxidoreductase NAD(P)H dehydrogenase, quinone 1 (NQO1) as the major target of dimeric naphthoquinones and proposed a mechanism of action that entailed induction of a futile redox cycling.

Results

Here, for the first time, we describe a direct physical interaction between the bromohydroxy dimeric naphthoquinone E6a and NQO1. Moreover, our studies reveal an extensive binding interface between E6a and the isoalloxazine ring of the flavin adenine dinucleotide (FAD) cofactor of NQO1 in addition to interactions with protein side chains in the active site. We also present biochemical evidence that dimeric naphthoquinones affect the redox state of the FAD cofactor of NQO1. Comparison of the mode of binding of E6a with those of other chemotherapeutics reveals unique characteristics of the interaction that can be leveraged in future drug optimization efforts.

Conclusion

The first structure of a dimeric naphthoquinone-NQO1 complex was reported, which can be used for design and synthesis of more potent next generation dimeric naphthoquinones to target NQO1 with higher affinity and specificity.

     

Interaction of tyrosine-phenol-lyase from Citrobacter intermedius with amino acids and their derivatives: factors determining the effectiveness of binding

The inhibition by L-amino acids and their derivatives of tyrosine phenol-lyase is investigated. Tyramine, alpha-phenylethylamine and tryptamine have no detectable inhibition effect and hence are weakly bonded by an active site. The aromatic amino acid amides are competitive inhibitors but do not manifest an enzymatic isotope exchange of alpha-proton in D2O. Free amino acids however are competitive inhibitors and in the majority of cases exchange alpha-proton. The presence of COOH-group is therefore an important feature which determines the binding efficiency and causes the "active" conformation of the amino acid-PLP complex labelising alpha-proton. In the absence of functional and bulky groups in the amino acid side chain the hydrophobicity is found to be the main factor determining the binding efficiency. For these amino acids a correlation exists between-RTlnKi and side chain hydrophobicity. The amino acids bearing the bulky groups, i. e. valine, leucine and isoleucine have reduced binding efficiency. Lysine and arginine bearing positively charged functional groups possess no inhibition effect. Aspartic and glutamic acids are anomalously strong inhibitors taking into consideration low hydrophobicity of their side chains. One can assume that the electrophilic group able to interact with the terminal COO- -group of aspartic and glutamic acids is located in the active site of tyrosine phenollyase.     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.     

Insensitivity to Abeta42-lowering nonsteroidal anti-inflammatory drugs and gamma-secretase inhibitors is common among aggressive presenilin-1 mutations

Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors.     

Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 1. Mechanism of inhibition by amiloride analogues

The mechanism by which terminal guanidino nitrogen substituted analogues of amiloride inhibit Na-Ca exchange in purified cardiac sarcolemmal membrane vesicles has been investigated. These inhibitors block both Nai-dependent Ca2+ uptake and Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange monitored in K+ is noncompetitive vs Ca2+ but competitive vs Na+. Substitution of sucrose for K+ results in mixed kinetics of inhibition vs Ca2+, suggesting a complex interaction between inhibitor and carrier under this condition. Amiloride derivatives also block two other modes of carrier action: Na-Na exchange is inhibited in a competitive fashion with Na+ and kinetics of Ca-Ca exchange inhibition are mixed vs Ca2+ in either sucrose or K+. However, Ca-Ca exchange inhibition can be alleviated by increasing K+ concentration. Dixon analyses of Na-Ca exchange block with mixtures of inhibitors suggest that these agents are interacting at more than one site. In addition, Hill plots of inhibition are biphasic with Hill coefficients of 1 and 2 at low and high inhibitor concentrations, respectively. These results indicate that amiloride derivatives are mechanism-based inhibitors that interact at two classes of substrate-binding sites on the carrier; at low concentration they bind preferentially to a site that is exclusive for Na+, while at higher concentration they also interact at a site that is common for Na+, Ca2+, and K+.     

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.

The class C serine beta-lactamase of Enterobacter cloacae P99 was inhibited by a series of aryl methylphosphonate monoester monoanions. The effectiveness of these inhibitors was promoted by an acylamido substituent on the methyl group and a good leaving group at phosphorus. The former preference suggests that noncovalent interaction of these inhibitors with the enzyme resembles that of substrates, while the latter suggests that nucleophilic displacement at phosphorus occurs as part of the inhibition mechanism. The truth of the latter proposition was confirmed by observation of release of 1 equiv of phenol concomitant with inhibition and of the presence of an equivalent amount of 14C-label on the enzyme after inhibition by a 14C-labeled phosphonate. The hydrolytically inert nature of the enzyme-inhibitor adduct, and its 31P chemical shift, suggested that O-phosphonylation of the enzyme had occurred. Although, by analogy with substrates, one might expect that the hydroxyl of the active site serine residue would be covalently modified by these inhibitors, successive alkali and acid treatment of the enzyme-inhibitor adduct generated no pyruvate. Instead, 1 equiv of lysinoalanine was found. This product was rationalized to arise through intramolecular capture by an adjacent lysine amine group of the dehydroalanine residue produced by alkali treatment of an O-phosphonylated serine residue. One equivalent of lysinoalanine was also produced by alkali treatment of the enzyme that had been inhibited by 6 beta-bromopenicillanic acid, a mechanism-based inhibitor known to acylate the hydroxyl group of the active site serine residue. It is therefore likely that the aryl phosphonates phosphonylate this residue. These compounds should be useful as beta-lactamase active site titrants and as sources of fresh insight into the chemical properties of the active site. The significant mechanistic features of the inhibition, in particular its strong leaving group dependence and the distinctive ability of the beta-lactamase active site to stabilize a dianionic transition state containing a pentacoordinated phosphorus, are discussed with respect to the active site structure. The comparison with phosph(or/on)yl inhibitors of serine proteinases is made, and the mechanism-based features of inhibition of serine hydrolases by phosph(on)ates are noted.     

Histone H3 peptides incorporating modified lysine residues as lysine-specific demethylase 1 inhibitors

Lysine-specific demethylase 1 (LSD1) is a flavin-dependent enzyme that removes methyl groups from mono- or dimethylated lysine residues at the fourth position of histone H3. We have previously reported several histone H3 peptides containing an LSD1 inactivator motif at Lys-4. In this study, histone H3 peptides having a trans-2-phenylcyclopropylamine (PCPA), a 2,5-dihydro-1H-pyrrole, and a 1,2,3,6-tetrahydropyridine moiety at Lys-4 were prepared along with related compounds possessing a shorter side chain at the fourth position. Enzymatic assays showed that PCPA peptides containing a longer side chain, which can react with FAD in the active site, are potent LSD1-selective inhibitors.     

Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Copper(I), copper(II) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus capsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron-sulphur centre of protein C with a secondary effect at the FAD centre when the copper(II):protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron-sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule. These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.     

New substrates for beta-lactam-recognizing enzymes: aryl malonamates

Aryl malonamates are demonstrated to be novel substrates of a broad range of beta-lactam-recognizing enzymes. These compounds are isomers of the aryl phenaceturates, which are well-known substrates of these enzymes, but the new compounds contain a retro-amide side chain. Several lines of evidence, including comparisons of steady-state kinetic parameters between enzymes and a detailed investigation of the methanolysis kinetics, solvent deuterium isotope effects, and pH-rate profile for turnover of a retro substrate by the Enterobacter cloacae P99 beta-lactamase, suggested that the new substrates are likely to be hydrolyzed by the same chemical mechanisms as "normal" substrates. Molecular modeling indicated that the retro-amide group fits snugly into the active site of the P99 beta-lactamase by hydrogen bonding to the conserved lysine-67 residue. The retro-amide side chain may represent a lead to novel mechanism-based and transition state analogue inhibitors.     

The discovery of fluoropyridine-based inhibitors of the Factor VIIa/TF complex

The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid side chain could lead to the formation of more potent inhibitors through interactions with the S1'/S2' pocket.     

Inhibitor-resistant class A beta-lactamases: consequences of the Ser130-to-Gly mutation seen in Apo and tazobactam structures of the SHV-1 variant

A bacterial response to the clinical use of class A beta-lactamase inhibitors such as tazobactam and clavulanic acid is the expression of variant beta-lactamases with weaker binding affinities for these mechanism-based inhibitors. Some of these inhibitor-resistant variants contain a glycine mutation at Ser130, a conserved active site residue known to be adventitiously involved in the inhibition mechanism. The crystallographic structure of a complex of tazobactam with the Ser130Gly variant of the class A SHV-1 beta-lactamase has been determined to 1.8 A resolution. Two reaction intermediates are observed. The primary intermediate is an acyclic species bound to the reactive Ser70. It is poorly primed for catalytic hydrolysis because its ester carbonyl group is completely displaced from the enzyme's oxyanion hole. A smaller fraction of the enzyme contains a Ser70-bound aldehyde resulting from hydrolytic loss of the triazoyl-sulfinyl amino acid moiety from the primary species. This first structure of a class A beta-lactamase lacking Ser130, the side chain of which functions in beta-lactam binding and possibly in catalysis, gives crystallographic evidence that the acylation step of beta-lactam turnover can occur without Ser130. Unexpectedly, the crystal structure of the uncomplexed Ser130Gly enzyme, also determined to 1.8 A resolution, shows that a critical Glu166-activated water molecule is missing from the catalytic site. Comparison of this uncomplexed variant with the wild-type structure reveals that Ser130 is required for orienting the side chain of Ser70 and ensuring the hydrogen bonding of Ser70 to both Lys73 and the catalytic water molecule.