The Amino-Acid Substituents of Dipeptide Substrates of Cathepsin C Can Determine the Rate-Limiting Steps of Catalysis

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• 作者：Jon K. Rubach ; Guanglei Cui ; Jessica L. Schneck ; Amy N. Taylor ; Baoguang Zhao ; Angela Smallwood ; Neysa Nevins ; David Wisnoski ; Sara H. Thrall ; Thomas D. Meek
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：September 25, 2012
• 年：2012
• 卷：51
• 期：38
• 页码：7551-7568
• 全文大小：602K
• 年卷期：v.51,no.38(September 25, 2012)
• ISSN：1520-4995

文摘

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for k_cat/K_a and k_cat values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for k_cat/K_a (1.6 卤 0.09 渭M^鈥? s^鈥?) and k_cat (255 卤 6 s^鈥?), an inverse sKIE on k_cat/K_a (^D(k_cat/K_a) = 0.6 卤 0.15), a modest, normal sKIE on k_cat (^Dk_cat = 1.6 卤 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s^鈥?). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (k_ac) and deacylation (k_dac) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) Biochemistry 47, 8697鈥?710), were found to be less active substrates compared to Gly-Arg-AMC, with respective k_cat/K_a values of 0.49 卤 0.07 渭M^鈥?聽s^鈥? and 5.3 卤 0.5 渭M^鈥?聽s^鈥?, and k_cat values of 28 卤 1 s^鈥? and 25 卤 0.5 s^鈥?. Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for k_cat/K_a (^D(k_cat/K_a) = 0.74 卤 0.05) and normal for k_cat (^Dk_cat = 2.3 卤 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s^鈥? and 390 s^鈥?, respectively), from which it was determined that k_ac k_dac k_cat. The third substrate type, Gly-Ile-AMC, gave very low values of k_cat/K_a (0.0015 卤 0.0001 渭M^鈥? s^鈥?) and k_cat (0.33 卤 0.02 s^鈥?), no sKIEs, (^D(k_cat/K_a) = 1.05 卤 0.5 and ^Dk_cat = 1.06 卤 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which k_dac > k_ac k_cat, and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P₁ position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases.