Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates.
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)
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
D002413:Cations, Divalent; D008670:Metals; D003035:Cobalt; D003300:Copper; D009532:Nickel; D000626:Aminopeptidases; D015032:Zinc