The site of acetylation of glyceraldehyde-3-phosphate dehydrogenase by p-nitrophenylacetate

The Amino Acid Sequence of a Hexapeptide Containing an Essential Sulfhydryl Group of Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase*

“…During the course of our investigations, Harris et al (1963) briefly described the amino acid sequence of a tryptic peptide of the enzyme labeled with radioactive iodoacetate or p-nitrophenyl acetate, which appears to contain the peptic peptide discussed in the present study. Cunningham and Schepman (1963) have isolated a tripeptide from p-nitrophenyl acetatelabeled enzyme which may be a component of the hexapeptide described here.…”

The Acetylation of Creatine Phosphokinase with ρ-Nitrophenyl Acetate*

“…Effect of Previous Treatment with Iodoacetic Acid upon the p-Nitrophenyl Acetate Reaction. Since two of the six sulfhydryl groups of creatine phosphokinase have been shown to react with iodoacetic acid with a resultant loss of enzymic activity (Mahowald et al, 1962;Watts et al, 1962) and a sulfhydryl group has been shown to be the site of acetylation by p-nitrophenyl acetate in glyceraldehyde 3-phosphate dehydrogenase (Cunningham and Schepman, 1963), it was of interest to study the effect of prior iodoacetic acid treatment upon the />nitrophenyl acetate reaction with creatine phosphokinase. Figure 2 shows the liberation of p-nitrophenol to be only slightly, if at all, affected by prior reaction of the enzyme (3.4 mg/ml) with iodoacetic acid at pH 7.0, in 0.30 m Tris-HCl-lO"5 m EDTA buffer containing 0.5 mM iodoacetic acid, for 67 min at 25°.…”

“…Experiments on the isolated peptide resulting from Pronase digestion indicated no loss of radioactivity under these conditions. The small loss observed in the whole digests could be due to oxidation of small amounts of 5-acetyl residues since Cunningham and Schepman (1963) have shown that performic oxidation of the 5-acetyl residues of [14C]acetylglyceraldehyde 3-phosphate dehydrogenase leads to complete loss of all acetyl groups under these same conditions. The stability of the great majority of the [14C]acetyl groups demonstrates that the site of the inhibitory acetylation of creatine phosphokinase is not a cysteine residue.…”

“…T X he limited acetylation by p-nitrophenyl acetate of a single serine residue of chymotrypsin (Hartley and Kilby, 1954; Balls and Wood, 1956; Oosterbaan et al, 1962) and three cysteine residues of glyceraldehyde 3-phosphate dehydrogenase (Park et al, 1961;Cunningham and Schepman, 1963) has proved of considerable interest with regard to the catalytic mechanisms of these enzymes.. These specific reactions of activated amino acid residues are dependent upon the maintenance of the native conformation of the enzymes and result in the inhibition of enzymic activity.…”

“…These reactive side-chain functions have been at least partly related to the chemical mechanisms of normal catalysis of these enzymes, leading to the suggestion that specific three-dimensional orientations of combinations of certain amino acid side chains produce centers of unusual chemical reactivity which participate in enzymatic catalysis (Cunningham, 1957; Cunningham and Schepman, 1963). Thus, it was of interest to determine if the inhibition of creatine phosphokinase (CPK)1 (creatine-ATP transphosphorylase, ATP-creatine phosphotransferase, E.C.…”