Identification of the essential cysteine residue in the active site of bovine pyruvate dehydrogenase.
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.
Ali
M S
MS
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Roche
T E
TE
Patel
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000494:pharmacology
D002250:Carbon Radioisotopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D003545:Cysteine; D005033:Ethylmaleimide; D013835:Thiamine Pyrophosphate