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 enzymes related to the serine peptidase subtilisin have recently been identified in eukaryotes. Herein is described and kinetically characterized a new type of processing enzyme, oligopeptidase B, which is encountered in the prokaryote Escherichia coli, and belongs to the prolyl oligopeptidase family of serine peptidase. The enzyme hydrolyzes the peptides at the carboxy end of dibasic sites by two orders of magnitude faster with respect to monobasic substrates. The kcat/K(m) is extremely high, 63 microM-1 s-1, for the substrate benzyloxycarbonyl-L-arginyl-L-arginyl-7-(4-methylcoumaryl)amide. The bell-shaped pH dependence of the rate constant is perturbed by some ionizing group(s). This effect is abolished at 1 M NaCl. In addition, high ionic strength inhibits the reaction considerably by increasing K(m), which is indicative of an electrostatic interaction between the arginyl residues and the enzymatic carboxy groups. In distinction from that found with most serine endopeptidases, kinetic deuterium isotope measurements with oligopeptidase B indicate that the rate-limiting step of the reaction is a physical step rather than a chemical one characterized by general acid/base catalysis. The present result will contribute to our understanding of the processing phenomena in prokaryotes, as well as in higher organisms.
Polgár
L
L
Institute of Enzymology, Hungarian Academy of Sciences, Budapest. polgar@hanga.enzim.hu
D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000737:chemistry*; D055672:Static Electricity; D013379:Substrate Specificity
D001426:Bacterial Proteins; D012697:Serine Endopeptidases; C090703:oligopeptidase B