Substrate-Dependent Inhibition Kinetics of an Active Site-Directed Inhibitor of ADAMTS-4 (Aggrecanase 1)

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• 作者：Arthur J. Wittwer ; Robert L. Hills ; Robert H. Keith ; Grace E. Munie ; Elizabeth C. Arner ; Charles P. Anglin ; Anne-Marie Malfait ; Micky D. Tortorella
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：May 29, 2007
• 年：2007
• 卷：46
• 期：21
• 页码：6393 - 6401
• 全文大小：173K
• 年卷期：v.46,no.21(May 29, 2007)
• ISSN：1520-4995

文摘

ADAMTS-4 (aggrecanase-1) is implicated in the breakdown of articular cartilage and is anattractive target for therapeutic intervention in arthritis. Cleavage of the native substrate, aggrecan, occursthrough exosite interactions and peptide sequence recognition. Although expected to be competitive withaggrecan, the hydroxamic acid, SC81956, demonstrated noncompetitive inhibition kinetics with a K_i of23 nM. The IC₅₀ of SC81956 did not change when aggrecan was varied from 12.8 to 200 nM (0.2-3.3times the apparent aggrecan K_m of 61 nM) but was shifted as expected for a competitive inhibitor whenincreasing levels of a low molecular weight peptide substrate were added to a fluorogenic peptide assaysystem. These observations are consistent with a model for aggrecan cleavage where substrate initiallybinds at an exosite, followed by binding of the appropriate peptide sequence at the active site. A peptide-competitive inhibitor could bind both free enzyme and initial substrate-enzyme exosite complex butwould be excluded by the final Michaelis complex. Noncompetitive appearing kinetics for such inhibitorsis predicted as long as the equilibrium between the two forms of enzyme-substrate complex significantlyfavors the initial exosite complex. In support, hydrolysis of a low molecular weight peptide substrate andits inhibition by SC81956 were unaffected by aggrecan concentrations substantially above the K_m. Theseobservations suggest that the apparent K_m for aggrecan cleavage predominately reflects the exositeinteraction. Consequently, the efficacy of active-site inhibitors of ADAMTS-4 will not be limited bycompetition with native substrate as predicted from the K_m determined by traditional kinetic models.