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 approximately 50-kDa extracellular metalloproteases (serralysins). The pH range of catalytic activity extends uniformly from 4 to greater than 10 (k(cat)/Km approximately 10(3) s(-1) M(-1), similar profile for k(cat)). 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 (Tyr216) that is crystallographically found ligated to the active-site Zn2+ of the uncomplexed enzyme must have a pKa of approximately 5. A chemical catalytic mechanism of proteolysis consistent with the kinetic data is proposed, in which Tyr216-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.
Mock
W L
WL
Department of Chemistry, University of Illinois at Chicago, 60607-7061, USA. wlmock@uic.edu
Yao
J
J
D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D013054:Spectrophotometry, Atomic
D008666:Metalloendopeptidases; C071288:astacin; C095666:serralysin