Kinetic characterization of the serralysins: A divergent catalytic mechanism pertaining to astacin-type metalloproteases

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 similar to 50-kDa extracellular metalloproteases (serralysins). The pH range of catalytic activity extends uniformly from 4 to greater than 10 (k(cat)/K-m similar to 10(3) s(-1) M-1, similar profile for k(cat)). Substrate analogue hydroxamic acid Cbz-Leu-Ala-NHOH competitively inhibits serralysin (K-i 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 pK(a) of similar to 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.