Crystal structures of dialkylglycine decarboxylase inhibitor complexes.

The crystal structures of four inhibitor complexes of dialkylglycine decarboxylase are reported. The enzyme does not undergo a domain closure, as does aspartate aminotransferase, upon inhibitor binding. Two active-site conformations have been observed in previous structures that differ in alkali metal ion content, and two active-site conformations have been shown to coexist in solution when a single type of metal ion is present. There is no indication of coexisting conformers in the structures reported here or in the previously reported structures, and the observed conformation is that expected based on the presence of potassium in the enzyme. Thus, although two active-site conformations coexist in solution, a single conformation, corresponding to the more active enzyme, predominates in the crystal. The structure of 1-aminocyclopropane-1-carboxylate bound in the active site shows the aldimine double bond to the pyridoxal phosphate cofactor to be fully out of the plane of the coenzyme ring, whereas the Calpha-CO2(-) bond lies close to it. This provides an explanation for the observed lack of decarboxylation reactivity with this amino acid. The carboxylate groups of both 1-aminocyclopropane-1-carboxylate and 5'-phosphopyridoxyl-2-methylalanine interact with Ser215 and Arg406 as previously proposed. This demonstrates structurally that alternative binding modes, which constitute substrate inhibition, occur in the decarboxylation half-reaction. The structures of d and l-cycloserine bound to the active-site show that the l-isomer is deprotonated at C(alpha), presumably by Lys272, while the d-isomer is not. This difference explains the approximately 3000-fold greater potency of the l versus the d-isomer as a competitive inhibitor of dialkylglycine decarboxylase.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus

A serine protease inhibitor was purified from plasma of the eastern oyster, Crassostrea virginica. The inhibitor is a 7609.6 Da protein consisting of 71 amino acids with 12 cysteine residues that are postulated to form 6 intra-chain disulfide bridges. Sequencing of the cloned cDNA identified an open reading frame encoding a polypeptide of 90 amino acids, with the 19 N-terminal amino acids forming a signal peptide. No sequence similarity with known proteins was found in sequence databases. The protein inhibited the serine proteases subtilisin A, trypsin and perkinsin, the major extracellular protease of the oyster protozoan parasite, Perkinsus marinus, in a slow binding manner. The mechanism of inhibition involves a rapid binding of inhibitor to the enzyme to form a weak enzyme-inhibitor complex followed by a slow isomerization to form a very tight binding enzyme-inhibitor complex. The overall dissociation constants K(i) with subtilisin A, perkinsin and trypsin were 0.29 nM, 13.7 nM and 17.7 nM, respectively. No inhibition of representatives of the other protease classes was detected. This is the first protein inhibitor of proteases identified from a bivalve mollusk and it represents a new protease inhibitor family. Its tight binding to subtilisin and perkinsin suggests it plays a role in the oyster host defense against P. marinus.     

Inhibition of factor Xa by a peptidyl-alpha-ketothiazole involves two steps. Evidence for a stabilizing conformational change.

Recently, peptidylketothiazoles have been shown to be potent inhibitors of proteases, but the details of the interaction have not yet been studied. In the work presented here, the interaction of factor Xa, a coagulation protease, with the transition state inhibitor BnSO(2)-D-Arg-Gly-Arg-ketothiazole (C921-78) is characterized. C921-78 is a tight and selective inhibitor of the coagulation protease factor Xa (K(d) = 14 pM). The hydrolytic activity of factor Xa was inhibited by C921-78 in a time-dependent manner. The rate-limiting step of the bimolecular combination of inhibitor and enzyme was competitive with the substrate. Conversely, the inhibitor could be displaced from the active site of the enzyme after exposure of the preformed complex to an excess of substrate or to the active site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CMK) in a slow reaction. The formation of the C921-78-factor Xa complex resulted in a 60% increase in the magnitude of the fluorescence emission spectrum. Rapid mixing of the enzyme and inhibitor produces a monophasic fluorescence increase, compatible with spectral transition in a single step. The rate constant for this reaction increased hyperbolically with the concentration of C921-78, but the amplitude remained constant. These results are consistent with the initial formation of an enzyme-inhibitor complex (EI), followed by a unimolecular conversion of EI to EI linked to a spectral transition. The rate constants of the isomerization provide an estimate of 300000-fold stabilization. Thus, the inhibition of factor Xa by C921-78 follows a mechanism similar to that described classically for slow tight binding inhibitors. However, the two steps of the reaction cannot be kinetically separated by the rapid equilibrium assumption, and therefore, the formation of EI is partially rate-limiting, too. The driving energy for the unusually fast isomerization step may result from the highly favorable interactions of the inhibitor in the primary binding site.     

Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease

The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.     

Slow tight binding inhibition of proteinase K by a proteinaceous inhibitor: conformational alterations responsible for conferring irreversibility to the enzyme-inhibitor complex

The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 +/- 0.5 x 10-5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 +/- 0.6 x 10-6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant k5 (9.2 +/- 1 x 10-3 s-1). The rate of dissociation of EI* (k6) was slower (4.5 +/- 0.5 x 10-5 s-1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 +/- 0.3 x 10-7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.     

Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue

Lavendustin-A was reported to be a potent tyrosine kinase inhibitor of the epidermal growth factor (EGF) receptor (Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshibi, K., Naganawa, H., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1989) J. Nat. Prod. 52, 1252-1257). Its inhibition kinetics was studied in detail using the baculovirus-expressed recombinant intracellular domain of the EGF receptor (EGFR-IC). Lavendustin-A (RG 14355) is a slow and tight binding inhibitor of the receptor tyrosine kinase. The pre-steady state kinetic analysis demonstrates that the inhibition corresponds to a two-step mechanism in which an initial enzyme-inhibitor complex (EI) is rapidly formed followed by a slow isomerization step to form a tight complex (EI*). The dissociation constant for the initial rapid forming complex is 370 nM, whereas the overall dissociation constant is estimated to be less than or equal to 1 nM. The difference between the two values is due to the tight binding nature of the inhibitor to the enzyme in EI*. The kinetic analysis using a preincubation protocol to pre-equilibrate the enzyme with the inhibitor in the presence of one substrate showed that Lavendustin-A is a hyperbolic mixed-type inhibitor with respect to both ATP and the peptide substrate, with a major effect on the binding affinities for both substrates. An analogue of Lavendustin-A (RG 14467) showed similar inhibition kinetics to that of Lavendustin-A. The results of the pre-steady state analysis are also consistent with the proposed two-step mechanism. The dissociation constant for the initial fast forming complex in this case is 3.4 microM, whereas the overall dissociation constant is estimated to be less than or equal to 30 nM. It is a partial (hyperbolic) competitive inhibitor with respect to ATP. Its inhibition is reduced to different extents by different peptide substrates, when the peptide is added to the enzyme simultaneously with the inhibitor. When studied with the least protective peptide, K1 (a peptide containing the major autophosphorylation site of the EGF receptor), RG 14467 acts as a hyperbolic noncompetitive inhibitor with respect to the peptide.     

Mechanism of Inhibition of beta-site amyloid precursor protein-cleaving enzyme (BACE) by a statine-based peptide

Inhibition of beta-site amyloid precursor protein-cleaving enzyme by a statine-based inhibitor has been studied using steady state and stopped-flow methods. A slow onset rate of inhibition has been observed under steady state conditions, and a K(i) of 22 nm has been derived using progress curves analysis. Simulation of stopped-flow protein fluorescence transients provided an estimate of the K(d) for initial inhibitor binding of 660 nm. A two-step inhibition mechanism is proposed, wherein slower "tightening up" of the initial encounter complex occurs. Two hypotheses have been proposed in the literature to address the nature of the slow step in the inhibition of aspartic proteases by peptidomimetic inhibitors: a conformational change related to the "flap" movement and displacement of a catalytic water. We compared substrate and inhibitor binding rates under pre-steady-state conditions. Both ligands are likely to cause flap movement, whereas no catalytic water replacement occurs during substrate binding. Our results suggest that both ligands bind to the enzyme at a rate significantly lower than the diffusion limit, but there are additional rate limitations involved in inhibitor binding, resulting in a k(on) of 3.5 x 10(4) m(-)1 s(-)1 for the inhibitor compared with 3.5 x 10(5) m(-)1 s(-)1 for the substrate. Even though specific intermediate formation steps might be different in the productive inhibitor and substrate binding to beta-site amyloid precursor protein-cleaving enzyme, a similar final optimized conformation is achieved in both cases, as judged by the comparable free energy changes (DeltaDeltaG of 2.01 versus 1.97 kcal/mol) going from the initial to the final enzyme-inhibitor or enzyme-substrate complexes.     

Inhibition of 1,4-beta-D-xylan xylanohydrolase by the specific aspartic protease inhibitor pepstatin: probing the two-step inhibition mechanism

This is the first report that describes the inhibition mechanism of xylanase from Thermomonospora sp. by pepstatin A, a specific inhibitor toward aspartic proteases. The kinetic analysis revealed competitive inhibition of xylanase by pepstatin A with an IC50 value 3.6 +/- 0.5 microm. The progress curves were time-depended, consistent with a two-step slow tight binding inhibition. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 15 +/- 1 x 10(-5) and 3.0 +/- 1 x 10(-8) s(-1), respectively. The Ki value for the formation of EI complex was 1.5 +/- 0.5 microm, whereas the overall inhibition constant Ki* was 28.0 +/- 1 nm. The conformational changes induced in Xyl I by pepstatin A were monitored by fluorescence spectroscopy, and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. Pepstatin A binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde-labeled xylanase. Our results revealed that the inactivation of xylanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis, and a model depicting the probable interaction between pepstatin A with xylanase has been proposed.     

Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations

Nonsteroidal antiinflammatory drugs (NSAIDs) block prostanoid biosynthesis by inhibiting prostaglandin H(2) synthase (EC 1.14.99.1). NSAIDs are either rapidly reversible competitive inhibitors or slow tight-binding inhibitors of this enzyme. These different modes of inhibition correlate with clinically important differences in isoform selectivity. Hypotheses have been advanced to explain the different inhibition kinetics, but no structural data have been available to test them. We present here crystal structures of prostaglandin H(2) synthase-1 in complex with the inhibitors ibuprofen, methyl flurbiprofen, flurbiprofen, and alclofenac at resolutions ranging from 2.6 to 2.75 A. These structures allow direct comparison of enzyme complexes with reversible competitive inhibitors (ibuprofen and methyl flurbiprofen) and slow tight-binding inhibitors (alclofenac and flurbiprofen). The four inhibitors bind to the same site and adopt similar conformations. In all four complexes, the enzyme structure is essentially unchanged, exhibiting only minimal differences in the inhibitor binding site. These results argue strongly against hypotheses that explain the difference between slow tight-binding and fast reversible competitive inhibition by invoking global conformational differences or different inhibitor binding sites. Instead, they suggest that the different apparent modes of NSAID binding may result from differences in the speed and efficiency with which inhibitors can perturb the hydrogen bonding network around Arg-120 and Tyr-355.     

Thiols as classical and slow-binding inhibitors of IMP-1 and other binuclear metallo-beta-lactamases

The inhibitory effect of a variety of thiol compounds on the function of binuclear metallo-beta-lactamases, with a particular focus on IMP-1 from Pseudomonas aeruginosa, has been investigated. Thiol inhibitors, depending on their structural features, fall into two categories, one in which inhibition at neutral pH was instantaneous and the other in which inhibition was time-dependent. While mercaptans with anionic substituents in the vicinity of their SH groups exhibited the former type of inhibition, neutral thiols appear to induce a slow, time-dependent isomerization of the initially formed EI complex to a tighter EI complex. Kinetic parameters describing the latter process were obtained by fitting progress curves of substrate hydrolysis using standard and numerical procedures. The failure of charged thiols to exhibit slow binding is suggested to be due to a rapid isomerization of the initial EI complex. Slow binding in the case of neutral thiols was observed only below pH 8. Studies on the pH dependence of catalysis by IMP-1 revealed that (i) enzyme inactivation at low pH is a slow process with presumably two groups with a pK(a) of approximately 5.2 in the protein being responsible for the loss of activity, (ii) inhibition by thiols is independent of pH between pH 5 and 9, and (iii) an apparent enhancement of the catalytic activity of IMP-1 by thiols occurs at pH <5. The last mentioned phenomenon is explained by a model in which mercaptans retard the proton-dependent isomerization of the enzyme. Studies on the thiol-mediated inhibition of the binuclear forms of Bacteroides fragilis (CcrA) and Bacillus cereus (BcII strain 5/B/6) metallo-beta-lactamase have revealed that while CcrA was instantaneously albeit moderately inhibited by mercaptans, BcII mimicked IMP-1 in its interaction with thiols. These differences are proposed to be due partly to the structural divergence of these proteins in the vicinity of Zn2.     

Potent mechanism-based inhibitors for matrix metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k(on) of 10(2) to 10(4) M(-1) s(-1) and the reversal of the process is slow (k(off) of 10(-3) to 10(-4) s(-1)). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.     

The kinetics of glucocorticoid binding to the soluble specific binding protein of mouse fibroblasts.

The kinetics of binding of glucocorticoids to the soluble, specific binding protein of mouse fibroblasts has been examined. The rate at which both potent and weak glucocorticoids achieve binding equilibrium is very slow. Second order rate constants of association range from 3 times 10-5 M- minus 1 min- minus 1 for cortisol to 6.7 times 10-5 M- minus 1 min- minus 1 for triamcinolone acetonide. Studies of the rates of binding at high steroid concentrations suggest that the slow rate of binding may be explained by a two-step mechanism. Active glucocorticoids, regardless of their potency, bind initially in a rapid manner to form a weak complex with the binding protein. The dissociation constant for the weak binding reaction is 0.87 times 10- minus 7 M for triamcinolone acetonide and 2.4 times 10- minus 7 M for cortisol. The weak binding complex becomes converted slowly to a tight complex. The first order rate constants for this conversion and the rate constants of dissociation from the tight complex have been determined for cortisol, dexamethasone and triamcinolone acetonide. The binding affinity of steroids of different biological potency is correlated with their rate of dissociation from this second tight binding state.     

The slow, tight binding of bestatin and amastatin to aminopeptidases

Bestatin reversibly inhibits Aeromonas aminopeptidase (EC 3.4.11.10) in a process that is remarkable for its unusual degree of time dependence. The binding of bestatin by both Aeromonas aminopeptidase and cytosolic leucine aminopeptidase (EC 3.4.11.1) is slow and tight, with Ki values (determined from rate constants) of 1.8 X 10(-8) and 5.8 X 10(-10) M, respectively. In contrast, microsomal aminopeptidase (EC 3.4.11.2) binds bestatin in a rapidly reversible process with a Ki value of 1.4 X 10(-6) M. Kinetic analysis of the slow inhibition observed is facilitated by the use of a variety of experimental treatments, primarily measurements made during pre-equilibrium; however, careful selection of conditions permits use also of steady state observations. When titrated with bestatin, 1 mol of cytosolic leucine aminopeptidase (containing 6 g atoms each of zinc and manganese) is rendered 80% inactive by 1 mol of inhibitor, thus suggesting that enzymatic activity depends on one active site/hexamer; titration of Aeromonas aminopeptidase by bestatin reveals a 1:1 stoichiometry. Amastatin inhibits all three aminopeptidases through the mechanism of slow, tight binding with Ki values ranging from 3.0 X 10(-8) to 2.5 X 10(-10) M. This behavior of microsomal aminopeptidase contrasts sharply with its rapidly reversible inhibition by bestatin. The slow, tight binding observed with five of the six aminopeptidase-inhibitor pairs investigated suggests the formation of a transition state analog complex between the enzyme and inhibitor. Physical evidence consistent with this possibility was provided by the observation that both bestatin and amastatin perturb the absorption spectrum of cobalt Aeromonas aminopeptidase.     

Chimerism reveals a role for the streptokinase Beta -domain in nonproteolytic active site formation,substrate, and inhibitor interactions

Streptokinase (SK) and staphylokinase form cofactor-enzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K(m) = 16.8 versus 15.2 microm(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.     

Crystal structures of unbound and aminooxyacetate-bound Escherichia coli gamma-aminobutyrate aminotransferase

The X-ray crystal structures of Escherichia coli gamma-aminobutyrate aminotransferase unbound and bound to the inhibitor aminooxyacetate are reported. The enzyme crystallizes from ammonium sulfate solutions in the P3(2)21 space group with a tetramer in the asymmetric unit. Diffraction data were collected to 2.4 A resolution for the unliganded enzyme and 1.9 A resolution for the aminooxyacetate complex. The overall structure of the enzyme is similar to those of other aminotransferase subgroup II enzymes. The ability of gamma-aminobutyrate aminotransferase to act on primary amine substrates (gamma-aminobutyrate) in the first half-reaction and alpha-amino acids in the second is proposed to be enabled by the presence of Glu211, whose side chain carboxylate alternates between interactions with Arg398 in the primary amine half-reaction and an alternative binding site in the alpha-amino acid half-reaction, in which Arg398 binds the substrate alpha-carboxylate. The specificity for a carboxylate group on the substrate side chain is due primarily to the presence of Arg141, but also requires substantial local main chain rearrangements relative to the structurally homologous enzyme dialkylglycine decarboxylase, which is specific for small alkyl side chains. No iron-sulfur cluster is found in the bacterial enzyme as was found in the pig enzyme [Storici, P., De Biase, D., Bossa, F., Bruno, S., Mozzarelli, A., Peneff, C., Silverman, R. B., and Schirmer, T. (2004) J. Biol. Chem. 279, 363-73.]. The binding of aminooxyacetate causes remarkably small changes in the active site structure, and no large domain movements are observed. Active site structure comparisons with pig gamma-aminobutyrate aminotransferase and dialkylglycine decarboxylase are discussed.     

Competitive inhibitors of Klebsiella aerogenes urease. Mechanisms of interaction with the nickel active site

We examined several compounds for their mechanisms of inhibition with the nickel-containing active site of homogeneous Klebsiella aerogenes urease. Thiolate anions competitively inhibit urease and directly interact with the metallocenter, as shown by the pH dependence of inhibition and by UV-visible absorbance spectroscopic studies. Cysteamine, which possesses a cationic beta-amino group, exhibited a high affinity for urease (Ki = 5 microM), whereas thiolates containing anionic carboxyl groups were uniformly poor inhibitors. Phosphate monoanion competitively inhibits a protonated form of urease with a pKa of less than 5. Both the thiolate and phosphate inhibition results are consistent with charge repulsion by an anionic group in the urease active site. Acetohydroxamic acid (AHA) was shown to be a slow-binding competitive inhibitor of urease. This compound forms an initial E.AHA complex which then undergoes a slow transformation to yield an E.AHA* complex; the overall dissociation constant of AHA is 2.6 microM. Phenylphosphorodiamidate, also shown to be a slow-binding competitive inhibitor, possesses an overall dissociation constant of 94 pM. The tight binding of phenylphosphorodiamidate was exploited to demonstrate the presence of two active sites per enzyme molecule. Urease contains 4 mol of nickel/mol enzyme, hence there are two nickel ions/catalytic unit. Each of the two slow-binding inhibitors are proposed to form complexes in which the inhibitor bridges the two active site nickel ions. The inhibition results obtained for K. aerogenes urease are compared with inhibition studies of other ureases and are interpreted in terms of a model for catalysis proposed for the jack bean enzyme (Dixon, N.E., Riddles, P.W., Gazzola, C., Blakely, R.L., and Zerner, B. (1980) Can. J. Biochem. 58, 1335-1344).     

A new class of potent, slowly reversible dehydropeptidase inhibitors

Dehydrodipeptide analogs whose scissile carboxamide has been replaced with a PO(OH)CH2 group have been found to be potent inhibitors of the zinc protease dehydrodipeptidase 1 (DHP-1, renal dipeptidase, EC 3.4.13.11). The best of these inhibitors, compound 25 (Ki = 0.52 nM), is two hundred times more potent than cilastatin 2 which is used clinically as a component of the broad-spectrum antibiotic combination Primaxin. Compound 25 is a tight binding inhibitor exhibiting slow binding kinetics with a remarkably slow off rate from DHP-1 (half life greater than 8 hours). The kinetics of its binding are consistent with a simple on-off mechanism whereas the less active D-enantiomer 26 appears to bind in an initial loose complex with the enzyme which slowly rearranges to a tighter complex (Ki = 83 nM).     

Possible role for water dissociation in the slow binding of phosphorus-containing transition-state-analogue inhibitors of thermolysin

A number of phosphonamidate and phosphonate tripeptide analogues have been studied as transition-state-analogue inhibitors of the zinc endopeptidase thermolysin. Those with the form Cbz-GlyP(Y)Leu-X [ZGP(Y)LX, X = NH2 or amino acid, Y = NH or O linkage] are potent (Ki = 9-760 nM for X = NH, 9-660 microM for X = O) but otherwise ordinary in their binding behavior, with second-order rate constants for association (kon) greater than 10(5) M-1 s-1. Those with the form Cbz-XP(Y)-Leu-Ala [ZXP(Y)LA,XP = alpha-substituted phosphorus amino acid analogue] are similarly potent (Ki for ZFPLA = 68 pM) but slow binding (kon less than or equal to 1300 M-1 s-1). Several kinetic mechanisms for slow binding behavior are considered, including two-step processes and those that require prior isomerization of inhibitor or enzyme to a rare form. The association rates of ZFPLA and ZFP(O)LA are first order in inhibitor concentration up to 1-2 mM, indicating that any loose complex along the binding pathway must have a dissociation constant above this value. The crystallographic investigation described in the preceding paper [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry (preceding paper in this issue)] identifies a specific water molecule in the active site that may hinder binding of the alpha-substituted inhibitors. The implication of this observation for a mechanism for slow binding is discussed.     

Elementary steps in the acquisition of Mn2+ by the fosfomycin resistance protein (FosA)

The fosfomycin resistance protein, FosA, catalyzes the Mn(2+)-dependent addition of glutathione to the antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid, rendering the antibiotic inactive. The enzyme is a homodimer of 16 kDa subunits, each of which contains a single mononuclear metal site. Stopped-flow absorbance/fluorescence spectrometry provides evidence suggesting a complex kinetic mechanism for the acquisition of Mn(2+) by apoFosA. The binding of Mn(H(2)O)(6)(2+) to apoFosA alters the UV absorption and intrinsic fluorescence characteristics of the protein sufficiently to provide sensitive spectroscopic probes of metal binding. The acquisition of metal is shown to be a multistep process involving rapid preequilibrium formation of an initial complex with release of approximately two protons (k(obsd) > or = 800 s(-1)). The initial complex either rapidly dissociates or forms an intermediate coordination complex (k > 300 s(-1)) with rapid isomerization (k > or = 20 s(-1)) to a set of tight protein-metal complexes. The observed bimolecular rate constant for formation of the intermediate coordination complex is 3 x 10(5) M(-1) s(-1). The release of Mn(2+) from the protein is slow (k approximately 10(-2) s(-1)). The kinetic results suggest a more complex chelate effect than is typically observed for metal binding to simple multidentate ligands. Although the addition of the substrate, fosfomycin, has no appreciable effect on the association kinetics of enzyme and metal, it significantly decreases the dissociation rate, suggesting that the substrate interacts directly with the metal center.