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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)

An extensive study of the requirements for effective binding of N-carboxyalkyl peptides to human stromelysin, collagenase, and to a lesser extent, gelatinase A has been investigated. These efforts afforded inhibitors generally in the 100-400 nM range for these matrix metalloproteinases. The most significant increase in potency was obtained with the introduction of a beta-phenylethyl group at the P1' position, suggesting a small hydrophobic channel into the S1' subsite of stromelysin. One particular compound, N-[1(R)-carboxyethyl]-alpha(S)-(2-phenylethyl)glycyl-L-leucine,N- phenylamide (79a), is relatively selective for rabbit stromelysin with a K(i) = 6.5 nM and may prove useful for elucidating the role of endogenously-produced stromelysin in lapine models of tissue degradation.