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.
Bartlett
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Department of Chemistry, University of California, Berkeley 94720.
Marlowe
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D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D063065:Organophosphonates / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013820:Thermolysin / Q000037:antagonists & inhibitors*; D014867:Water
D009842:Oligopeptides; D063065:Organophosphonates; D014867:Water; D013820:Thermolysin