Bromopyruvate as an affinity label for Baker's yeast flavocytochrome b2. Identification of an active-site cysteine and characterization of some cysteine peptides.

Abstract

It was previously reported that bromopyruvate behaves as an active-site-directed reagent for flavocytochrome b2 [Mulet and Lederer (1977) Eur. J. Biochem. 73, 443-447], but that some unspecific labeling also took place [Alliel, Mulet, and Lederer (1980) Eur. J. Biochem. 105, 343-351]. In this work, radioactive peptides were purified after labeling the enzyme with bromo[2-14C]pyruvate. Direct proteolysis of the labeled enzyme led to a multiplicity of labeled peptides, due to incomplete proteolysis. Four of them were characterized, corresponding to two unique cysteine residues. Cyanogen bromide cleavage of the labeled protein, followed by enzymatic digestion, led to the isolation of peptides corresponding to four cysteines, including the two previously identified ones. Comparison of the specific radioactivity of the various labeled peptides lead us to the conclusion that the active-site cysteine must be the one present in the 85-residue cyanogen bromide peptide alpha CB3. The sequence around that cysteine is Ala-Ser-Cys-Ser-Pro-Gln-Gln-Ile-Ile-Glu-Ala-Ala-.