Glyceraldehyde Phosphate Dehydrogenase of Escherichia coli

The molecular weight of the Escherichia coli glyceraldehyde phosphate dehydrogenase subunit in 5 m guanidine hydrochloride containing 0.01 m dithiothreitol was determined by high speed sedimentation equilibrium to be 35,000. Amino acid composition of the protein, taken in conjunction with the number of peptides obtained after tryptic digestion, indicated that the subunits of the enzyme were very probably identical, each containing 12 arginine and 26 lysine residues. NAD+ content of the protein, measured both by spectrophotometric and enzymatic methods, was found to be very low (l0.1 mole of NAD+ per 35,000 g of protein), although capacity to strongly bind NAD+ was intact. The enzyme could be crystallized but only after addition of NAD+ to apoprotein. One cysteine residue per subunit was carboxymethylated with resultant enzymatic inactivation when native enzyme was reacted with iodoacetic acid; similarly there was one sulfhydryl group per subunit which was rapidly reactive with 5,5'-dithiobis(2-nitrobenzoic acid). Under protein denaturing conditions a total of 3 cysteine residues per subunit were detected although analyses of performic acid-oxidized protein indicated four cysteic acids per subunit. Dehydrogenase activity of the enzyme was markedly sulfhydryl dependent; there was l25% of maximal activity if sulfhydryl compounds were not included in assay mixtures. Esterase activity (with p-nitrophenyl acetate as substrate) was inhibited by AMP, ADP, and ATP but not by NAD+.