Abstract
Pyruvate dehydrogenase (E1), the first catalytic component of the bovine pyruvate dehydrogenase complex, is composed of two nonidentical subunits in a tetrameric alpha 2 beta 2 form. The sulfhydryl-specific reagent N-ethylmaleimide (NEM) was used to identify the reactivities and function of cysteinyl residues and subsequent identification of these residues in the active site of bovine E1. Treatment of E1 with 0.2 mM NEM resulted in loss (90%) of enzymatic activity; the inactivation followed 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. To identify the reactive cysteinyl residues in the active site region, the nonessential SH groups in E1 were first modified with NEM in the presence of TPP and pyruvate. After quenching with dithiothreitol and removal of the substrate and cofactor by dialysis, the modified E1 was treated with [14C]NEM to label the exposed cysteinyl residue(s) in or near the active site region. The data indicate that NEM reacted in the active site region of the E1 component with a stoichiometry of 2 mol of [14C]NEM bound per mol of E1 tetramer. The initial rapid labeling of E1 with [14C]NEM established that incorporation was predominantly into the alpha subunit. A single radiolabeled peptide was isolated following V8 protease digestion of radiolabeled E1 by [14C]NEM. Sequence analysis of the labeled peptide derived from bovine E1 demonstrated that the labeled cysteinyl residue was equivalent to Cys-62 in the alpha subunit (mature form) of human E1.
Highlights
From the $Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and the §Department of Biochemistry, Kansas State University, Manhattan, Kansas66506
The available eviwas almost entirelyprevented by thiamin pyrophos- dence suggests thaEt la plays a crucial role in catalysis (Roche phate (TPP)and pyruvate; protectionis probably due to and Reed, 1972; Stepp and Reed1,9851, and E1P is involved in formation of the hydroxyethylidene-TPP intermediate. the binding of E l to the structural core of dihydrolipoamide
The involvement of cysteine residues in the catalytic function of bovine E l was indicated by the inactivation of the enzyme by the thiol-specific reagent NEM
Summary
Tnfluoroacetic acid and V8 protease were from Pierce Chemical Co. [1-l4C1NEM(38.7 mCi/mmol)from Du Pont-NewEngland Nuclear was diluted with unlabeled NEM and stored at -20 “C. Assay o f E l Activity-Bovine kidney PDC was purified as described previously (Roche and Cate, 1977).The PDC components were resolved. Site Active of Pyruvate Dehydrogenase according to established procedures (Linent al., 1972; Roche and Cate, 1977). Reconstitution assays were conducted wiathn excess of E 2 a n d E 3 added together withEl and the overallPDC activity was measured (Roche and Reed, 1972). Protein concentrations were determined spectrophotometrically usingthe Bio-Rad protein assay reagent withbovine
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