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The reactivity of the sulfhydryl groups of leucine aminopeptidase from bovine eye lens has been studied by carboxymethylation with iodoacetate of the native enzyme (Zn2+-Zn2+), of the enzyme activated with magnesium (Zn+-Mg2+) or manganese (Zn2+-Mn2+), the enzyme incubated with cobalt (CO2+-Co2+), and the metal-free apoenzyme. The reactivity of the sulfhydryl groups was also determined in the presence of denaturing agents with or without reducing agents. All seven half-cystines per leucine aminopeptidase subunit are in the sulfhydryl form. In the native and the Zn2+-Mn2+ enzyme, only one of the cysteines (residue 344) reacts readily with iodoacetate. In the Zn2+-Mg2+ enzyme, two cysteines react (residues 344 and 412) and in the Co2+-incubated enzyme only one (residue 412). The metal-free apoenzyme has at least three reactive cysteines (residues 344, 412, and 429). The data suggest that sulfhydryl groups are involved in the binding of metal ions.

The amino acid sequence of bovine eye lens leucine aminopeptidase has been determined. Cyanogen bromide fragments, the COOH-terminal hydroxylamine fragment, and a large fragment obtained by digestion with Staphylococcus aureus protease were isolated from reduced and S-alkylated leucine aminopeptidase. The amino acid sequences of these fragments were determined by automated sequence analysis, by manual direct Edman degradation, and by the dansyl-Edman technique. Overlapping peptides were obtained by tryptic digestion of the S-alkylated protein or the citraconylated S-alkylated protein. The polypeptide chain of leucine aminopeptidase comprises 478 residues, corresponding to a molecular weight of 51,691. No significant sequence homology with any other published protein primary structure could be detected. This is the first report of a complete amino acid sequence of an enzyme belonging to the class of two metal peptidases.