A POTENTIAL PROCESSING ENZYME IN PROKARYOTES - OLIGOPEPTIDASE-B, A NEW-TYPE OF SERINE PEPTIDASE

Basic amino acid pairs in polypeptides represent important markers for processing enzymes to produce biologically active products, Such enzy mes related to the serine peptidase subtilisin have recently been iden tified in eukaryotes, Herein is described and kinetically characterize d a new type of processing enzyme, oligopeptidase B, which is encounte red in the prokaryote Escherichia coli, and belongs to the prolyl olig opeptidase family of serine peptidases. The enzyme hydrolyzes the pept ides at the carboxy end of dibasic sites by two orders of magnitude fa ster with respect to monobasic substrates, The k(cat)/K-m is extremely high, 63 mu M-1 s(-1), for the substrate nyl-L-arginyl-L-arginyl-7-(4 -methylcoumaryl)amide. The bell-shaped pH dependence of the rate const ant is perturbed by some ionizing group(s), This effect is abolished a t I M NaCl. In addition, high ionic strength inhibits the reaction con siderably by increasing K-m, which is indicative of an electrostatic i nteraction between the arginyl residues and the enzymatic carboxy grou ps, In distinction from that found with most serine endopeptidases, ki netic deuterium isotope measurements with oligopeptidase B indicate th at the rate-limiting step of the reaction is a physical step rather th an a chemical one characterized by general acid/base catalysis, The pr esent result will contribute to our understanding of the processing ph enomena in prokaryotes, as well as in higher organisms. (C) 1997 Wiley -Liss, Inc.