Ketone-substrate analogues of Clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors

Mookhtiar, Kasim A.; Grobelny, Damian; Galardy, Richard E.; Wart, Harold E. Van

doi:10.1021/bi00412a016

Cited by 14 publications

(8 citation statements)

References 34 publications

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“…Substrateanalogue 'carbonyl hydrate' [HO-C(R2)-OH] complexes have also been examined, for carboxypeptidase A, with crystallographic observation of similarly variable metal-ion coordination involving this moiety [25,31]. In the latter cases it seems likely from the pH-dependence of K, [32] that the 'gem-diol' has its liganded oxygen deprotonated in the complex, i.e. the structures are simply hemiacetals formally derived from the addition of H-OZn(Enz) across the aldehyde or ketone group of the inhibitor.…”

Section: Discussionmentioning

confidence: 99%

Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis

Mock

Aksamawati

1994

Biochemical Journal

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Competitive inhibition as a function of pH for the metalloendoprotease thermolysin by derivatives of L-alpha-(2-hydroxyphenyl)benzenepropanoyl-L- tryptophanylglycylglycine exhibits a diagnostic bell shape. Binding is maximal between two pKa values: on the acidic limb the apparent Ki value is regulated by an unchanging enzymic ionization (pKa 5.3) which is also seen in the substrate-hydrolysis kinetics (kcat/Km), whereas the alkaline limb for inhibition varies and depends specifically on the pKa of the phenolic group in the inhibitor. Although it should be the phenolate form of the inhibitor that co-ordinates more efficiently to the active-site Zn2+, the apparent Ki shifts from pH-independent at pH values immediately below the inhibitor's pKa to progressively weaker binding at higher pH. This is explained by an anomalous acidity for the exchangeable solvent molecule that is attached to enzymic Zn2+ in the absence of substrate or inhibitor. Since OH- cannot be displaced from the enzyme as readily as H2O, a compensating pKa of 5.3 possessed by Zn(2+)-bound water rationalizes the binding characteristics, yielding the level pH profile exhibited at intermediate pH values. Recognition of the implicit heightened Lewis acidity of the metal ion in thermolysin leads to a revision of the mechanism of catalysis. The substrate amide bond becomes activated for hydrolysis by carbonyl-group co-ordination to the especially acidic Zn2+ ion (completely displacing the H2O/OH- species otherwise bound). The imidazole group of enzymic residue His-231, also discerned in the pH profile for kcat/Km from its pKa of 8, provides general-base assistance for hydration of the activated scissile linkage in the first committed step of catalysis. Additional evidence from inhibition patterns shows how substrate-binding energy may be employed in this scheme to promote hydrolysis of peptides by thermolysin.

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Section: Discussionmentioning

confidence: 99%

Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis

Mock

Aksamawati

1994

Biochemical Journal

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“…In addition to the residues involved in catalysis, the active site contains a certain number of residues responsible for substrate recognition which may vary between individual enzymes (Bouvier et al, 1989;Jongeneel et al, 1989), leading to the differences observed for substrate specificity. An important aspect to consider in designing inhibitors is the characterization of the subsite specificity of a given enzyme, in order to define the best peptide sequence to which the zinc ligand will be linked (Chu & Orlowski, 1984;Vencill et al, 1985;Orlowski et al, 1988;Mookhtiar et al, 1988). In this regard, the precise characterization of the substrate specificity of the Leishmania surface protease still requires more information.…”

Section: Discussionmentioning

confidence: 99%

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania

Bouvier¹,

Schneider²,

Etges³

et al. 1990

Biochemistry

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The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

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“…While usually invoked as a description of the design process, TS analogy can be confirmed by a correlation between the K{ values of a series of inhibitors and the Km/fccat values of the corresponding substrates, subject to certain qualifications (Wolfenden, 1976;Thompson, 1973;Bartlett & Marlowe, 1983). This correlation has been demonstrated most extensively for peptidases, and for the zinc peptidases in particular (Bartlett & Marlowe, 1983;Mookthiar et al, 1988;Hanson et al, 1989), because these enzymes permit considerable substrate variation and consequently a broad range of Km/kCil values.…”

mentioning

confidence: 88%

Transition-state characterization: a new approach combining inhibitor analogs and variation in enzyme structure

Phillips¹,

Kaplan²,

Rutter³

et al. 1992

Biochemistry

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A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.

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Ketone-substrate analogues of Clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors

Cited by 14 publications

References 34 publications

Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis

Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania

Transition-state characterization: a new approach combining inhibitor analogs and variation in enzyme structure

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