title: Spectral and kinetic studies of metal-substituted Aeromonas aminopeptidase: nonidentical, interacting metal-binding sites.
abstract: Apoenzyme prepared by removal of the 2 mol of Zn2+/mol from Aeromonas aminopeptidase is inactive. Addition of Zn2+ reactivates it completely, and reconstitution with Co2+, Ni2+, or Cu2+ results in a 5.0-, 9.8-, and 10-fold more active enzyme than native aminopeptidase, respectively. Equilibrium dialysis and spectral titration experiments with Co2+ confirm the stoichiometry of 2 mol of metal/mol. The addition of only 1 mol of metal/mol completely restores activity characteristic of the particular metal. Interaction between the two sites, however, causes hyperactivation; thus, addition of 1 mol of Zn2+/mol subsequent to 1 mol of Co2+, Ni2+, or Cu2+ per mole increases activity 3.2-, 42-, or 59-fold, respectively. The cobalt absorption spectrum has a peak of 527 nm with a molar absorptivity of 53 M-1 cm-1 for 1 mol of cobalt/mol, which increases to 82 M-1 cm-1 for a second cobalt atom and is unchanged by further addition of Co2+. Circular dichroic (CD) and magnetic CD spectra indicate that the first Co2+ binding site is tetrahedral-like and that the second is octahedral-like. Stoichiometric quantities of 1-butylboronic acid, a transition-state analogue inhibitor of the enzyme [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322], profoundly affects absorption, CD, and MCD spectra, but n-valeramide, a substrate analogue inhibitor, has no effect. These findings suggest that the tetrahedral-like site is catalytic and the other octahedral-like site is regulatory or structural.
mesh_terms: D000333:Aeromonas / Q000201:enzymology*; D000626:Aminopeptidases / Q000378:metabolism*; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry
chemical_list: D001051:Apoenzymes; D002413:Cations, Divalent; D008667:Metalloproteins; D000626:Aminopeptidases