Enzymatic Characterization of the N-Acetylation of 3-Phosphoglyceraldehyde Dehydrogenase by Acetyl Phosphate

Acetyl phosphate or p -nitrophenyl acetate acetylates a specific cysteine residue in the active center of 3-phosphoglyceraldehyde dehydrogenase crystallized from rabbit muscle. This reaction occurs more easily with the enzyme from rabbit than from yeast. At pH 7.0 and 0° the cysteine residue is readily acetylated and forms a common intermediate in the dehydrogenase, transferase, and esterase reactions. On warming or raising the pH to 8.5, the acetyl groups migrate from the cysteine to a specific lysine moiety by a S—N transfer reaction. Three to 4 cysteine or lysine residues can be acetylated per molecule of rabbit muscle dehydrogenase (mol wt 140,000). The distribution of the acetyl group between these 2 residues is affected by the pH, substrate concentration, and time of incubation. A more specifically and completely labeled N -acetyl enzyme can be prepared with acetyl phosphate than with p -nitrophenyl acetate. N -Acetylation of the enzyme impairs DPN binding and, depending on the assay conditions, produces varying degrees of inhibition of the dehydrogenase activity. On the other hand, DPN inhibits the N -acetylation of the enzyme. When DPN is added to the N -acetylated dehydrogenase, the coenzyme protects against progressive inactivation of the protein with time.