Hyun Ik Park and Li-June Ming*

Department of Chemistry and
Institute for Biomolecular Science
University of South Florida
Tampa, Florida 33620-5250

Abstract

 Serralysin is a bacterial endopeptidase which has been considered a virulence factor that causes tissue damage and anaphylactic response.  It is a member of metalloendopeptidase super-family containing a coordinated Tyr that is unique only to this family of Zn enzymes.  The coordinated Tyr in this endopeptidase family has been proposed to play an important role in the action of the enzyme.  Several metal-substituted derivatives of serralysin (including Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Cd²⁺ derivatives) are found to exhibit significant activities to different extents.  Particularly, the Co- and Cu-substituted derivatives exhibit much higher activities than the native serralysin toward the hydrolysis of benzoyl-Arg-p-nitroanilide, i.e., 60 and 37 times higher in kcat and 21 and 25 times in k_cat/K_m, respectively.  Such remarkably high activities of metal-substituted derivatives compared to that of the native enzyme are rare in the literature, reflecting the uniqueness of this enzyme among all Zn enzymes.  The significantly different kcat yet similar Km values among the several derivatives suggest that the metal center is involved in catalysis, but not necessarily in the binding of the substrate; whereas the dramatically different inhibitor constants of Arg-hydroxamate binding to the derivatives indicates direct binding of this inhibitor to the metal center.  The activity-pH profiles of serralysin and its Co²⁺ and Cu²⁺ derivatives and the optical-pH profile of Cu-serralysin have been obtained.  The decrease in activity at higher pHs was found to be associated with a dramatic increase in the Tyr-to-Cu²⁺ charge transfer transitions, which indicate that the binding of Tyr216 to the metal is inhibitory.  A metal-centered mechanism is proposed for serralysin catalysis based on the results presented here, which revises the general-base mechanism previously proposed.  In the mechanism proposed here, the detachment of the coordinated Tyr is considered an essential step during the formation of the transition state.

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