Studies on the kinetic and chemical mechanism of inhibition of stromelysin by an N-(carboxyalkyl)dipeptide.
We have investigated the inhibition of the human matrix metalloproteinase stromelysin (SLN) by the N-(carboxyalkyl)dipeptide Ala[N]hPhe-Leu-anilide and find that it is a competitive, slow-binding inhibitor of this enzyme with Ki = 3 x 10(-8) M (pH 6.0, 25 degrees C). The dependence of k(obs), the observed first-order rate constant for the approach to steady state, on Ala[N]hPhe-Leu-anilide concentrations less than 10(-5) M is linear and suggests a simple, one-step mechanism with kon = 3.4 x 10(4) M-1 s-1 and k(off) = 1.2 x 10(-3) s-1 (pH 6.0, 25 degrees C). Using rapid kinetic techniques, we extended the concentration range of Ala[N]hPhe-Leu-anilide to 2 x 10(-3) M and found that the [Ala[N]hPhe-Leu-anilide] dependence of K(obs) suggests saturation kinetics with a Ki' near 5 x 10(-4) M. Detailed analysis of these data reveal that the dependence of k(obs) on [Ala[N]hPhe-Leu-anilide] is, in fact, sigmoidal. To probe the chemical mechanism of inhibition, we determined pH and temperature dependencies and solvent deuterium isotope effects. For k(on), delta H not equal to = 12.4 kcal/mol and -T delta S not equal to = 6.2 kcal/mol (T = 298 K; [I]steady-state = 10(-6) M), while for k(off), delta H not equal to = 12.5 kcal/mol and -T delta S not equal to = 8.9 kcal/mol (T = 298 K). pH dependencies of the kinetic parameters for inhibition are complex but reflect greater potency at lower pH and suggest a mechanism involving the same active-site groups that are involved in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Izquierdo-Martin
M
M
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
Chapman
K T
KT
Hagmann
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Stein
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RL
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003903:Deuterium; D004151:Dipeptides / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D013696:Temperature; D013816:Thermodynamics
D004151:Dipeptides; D003903:Deuterium; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3