Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

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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., and 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 suceptible 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-leucin-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 ocupies, and, most strongly, by the substrate. The presence of Zn2+ in the first site generally results in high Km values in comparison with the other metalloenzymes and produces high kcat values toward both substrates with branched side chains, whereas Cu2+ in the first site yields low Km values with the two poorer substrates. A time dependence of activation occurs with metalloenzymes that have Cu2+ in the first site and another metal ion in the second binding site, but was not observed for any other combination of ions tested.