title: Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates.
abstract: Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000333:Aeromonas / Q000201:enzymology*; D000626:Aminopeptidases / Q000378:metabolism*; D001665:Binding Sites; D002413:Cations, Divalent; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology*; D009532:Nickel / Q000494:pharmacology; D011485:Protein Binding; D015032:Zinc / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D008670:Metals; D003035:Cobalt; D003300:Copper; D009532:Nickel; D000626:Aminopeptidases; D015032:Zinc