Kinetic Characterization of the Serralysins: A Divergent Catalytic Mechanism Pertaining to Astacin-Type Metalloproteases
- 1 April 1997
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 36 (16) , 4949-4958
- https://doi.org/10.1021/bi963149p
Abstract
Substrates HO2CCH2CH2CO- and HOCH2CHOHCHOHCO-Phe-Leu-Ala-5-nitro-2-pyridinamide are cleaved efficiently at the acylarenamide linkage, with a convenient spectrophotometric assay, by the Serratia and Pseudomonas ∼50-kDa extracellular metalloproteases (serralysins). The pH range of catalytic activity extends uniformly from 4 to greater than 10 (kcat/Km ∼103 s-1 M-1, similar profile for kcat). Substrate analogue hydroxamic acid Cbz-Leu-Ala-NHOH competitively inhibits serralysin (Ki 0.04 mM), with a pH dependence indicating that either a displaced metal-bound H2O or a similarly motile enzymic phenol residue (Tyr 216) that is crystallographically found ligated to the active-site Zn2+ of the uncomplexed enzyme must have a pKa of ∼5. A chemical catalytic mechanism of proteolysis consistent with the kinetic data is proposed, in which Tyr 216-ArO-, in the course of being released from the active-site metal ion, deprotonates a water molecule attacking the Zn2+-activated substrate linkage, leading to a metal-coordinated tetrahedral oxyanion adduct that subsequently fragments.Keywords
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