| Porcine kidney leucine aminopeptidase ((alpha)-amino-acyl peptide hydrolase, EC 3.4.11.1) has been purified by affinity chromatography over L-leucyglycyl-AH-Sepharose. The purified enzyme contains 1 gram atom per subunit of catalytically essential Zn('2+). Activation by Mg('2+) or Mn('2+), and inhibition by Cu('2+), Ni('2+), Zn('2+) or Hg('2+) is due to the binding of these ions to a second, regulatory site on each subunit. The catalytic pathway for the hydrolysis of peptides by metalloleucine aminopeptidases has been investigated in cryosolvents at subzero temperatures. The enzyme is very stable and is not denatured at 23(DEGREES)C in the presence of 50% v/v methanol, ethanol, dimethylsulfoxide or dimethylformamide. The effect of cosolvent concentration at 23(DEGREES)C is to decrease k(,cat) linearly and increase K(,M) exponentially. Arrhenius plots for peptide hydrolysis in the absence or presence of methanol give activation energies that agree favorably. All evidence shows that methanol does not perturb the catalytic pathway and is, therefore, a suitable cryosolvent for investigations at subzero temperatures. Direct visualization of E(.)S intermediates formed during the hydrolysis of L-Leu-Gly-NHNH-Dns by various {(LAP)Zn(,6)Me(,6)} in 50% v/v methanol, 0.1 M KCl, 1 mM Me('2+), 10 mM Tris, pH* 9.0 at subzero temperature has been carried out using the fluorescence energy transfer technique. Results show that the interconversion of (E(.)S)(,1) and (E(.)S)(,2) is detectable at -30(DEGREES)C or lower. A minimal reaction pathway that includes these two intermediates has been proposed. The regulating metal ion exerts a marked influence on both the steady-state and pre-steady-state kinetic parameters for this pathway. |
