Leucine Aminopeptidase (Bovine Lens)

Spark emission and atomic absorption spectroscopy of crystalline leucine aminopeptidase (bovine lens) (EC 3.4.1.1) shows the presence of 2 zinc atoms per subunit molecular weight of 54,000 (12 zinc atoms per oligomer of 320,000). Removal of zinc by dialysis yields a zinc-free product with no enzymatic activity which upon readdition of Zn2+, regains full activity with the concomitant binding of 2 zinc atoms per subunit. Addition of C&, Ca2+, Mg2+, or Mn*f to the zinc-free enzyme does not result in stoichiometric binding of these ions or in significant regain in activity. Incubation of the enzyme containing 2 zinc atoms with various concentrations of Zn2+ and Mg2+ or Zn2+ and Mn2+ shows (a) that there is a competition between the ions for one binding site, (b) that the ratio of dissociation constants for this site is KMc: K,, = 150 at pH 9.5 and KM,,: K,, = 35 at pH 8.5, and (c) that a maximum of 1 mole of Mg2+ or Mn2+ is bound per 54,000 g of leucine aminopeptidase. The results suggest that leucine aminopeptidase has two metal binding sites: a specificity site that must be occupied by zinc in order to have an active enzyme and an activation site which can be occupied by Zn2+, Mg2+, or Mn2+ to give zinc-zinc, zinc-magnesium, or zinc-manganese enzymes with different specific activities. The nature of the ion occupying the activation site has a pronounced effect on the maximum velocity of the reaction and a minor effect on the Michaelis constant. A new amino acid analysis of the lens leucine aminopeptidase is presented which accounts for all of the total nitrogen and dry weight of the enzyme in terms of amino acid residues and which indicates that the enzyme contains eight halfcystines per 54,000-dalton subunit. Six of these are in the sulfhydryl form, but only one of these is available for reaction in the native enzyme. This reactive sulfhydryl is not needed for enzymatic activity. On a dry weight basis the absorbance of a 1% solution of leucine aminopeptidase at neutral pH in a l-cm cuvette at 280 nm is 10. The partial specific volume as calculated from the amino acid composition is 0.74.