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., 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.