Sulfhydryl content of bovine eye lens leucine aminopeptidase. Determination of the reactivity of the sulfhydryl groups of the zinc metalloenzyme, of the enzyme activated by Mg2+, Mn2+, and Co2+, and of the metal-free apoenzyme.

Abstract

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.