IDENTIFICATION OF THE ESSENTIAL CYSTEINE RESIDUE IN THE ACTIVE-SITE OF BOVINE PYRUVATE-DEHYDROGENASE

Pyruvate dehydrogenase (E1), the first catalytic component of the bovi ne pyruvate dehydrogenase complex, is composed of two nonidentical sub units in a tetrameric alpha2beta2 form. The sulfhydryl-specific reagen t N-ethylmaleimide (NEM) was used to identify the reactivities and fun ction of cysteinyl residues and subsequent identification of these res idues in the active site of bovine E1. Treatment of E1 with 0.2 mM NEM resulted in loss (90%) of enzymatic activity; the inactivation follow ed bimolecular reaction kinetics. The inactivation was almost entirely prevented by thiamin pyrophosphate (TPP) and pyruvate; protection is probably due to formation of the hydroxyethylidene-TPP intermediate. T o identify the reactive cysteinyl residues in the active site region, the nonessential SH groups in E1 were first modified with NEM in the p resence of TPP and pyruvate. After quenching with dithiothreitol and r emoval of the substrate and cofactor by dialysis, the modified E1 was treated with [C-14]NEM to label the exposed cysteinyl residue(s) in or near the active site region. The data indicate that NEM reacted in th e active site region of the E1 component with a stoichiometry of 2 mol of [C-14]NEM bound per mol of E1 tetramer. The initial rapid labeling of E1 with [C-14]NEM established that incorportaion was predominantly into the alpha subunit. A single radiolabeled peptide was isolated fo llowing V8 protease digestion of radiolabeled E1 by [C-14]NEM. Sequenc e analysis of the labeled peptide derived from bovine E1 demonstrated that the labeled cysteinyl residue was equivalent to Cys-62 in the alp ha subunit (mature form) of human E1.