An Essential Histidine in the Catalytic Activities of 3-Phosphoglyceraldehyde Dehydrogenase

Abstract

Abstract Photooxidation of 3-phosphoglyceraldehyde dehydrogenase in the presence of rose bengal did not effect the rate or extent of acetylation of the enzyme with p-nitrophenyl acetate; however, the rate of deacetylation of the S-acetyl-enzyme was selectively inhibited by 50 to 60%. Amino acid analysis of the photooxidized enzyme showed about 1 histidine residue per monomer was destroyed. No other changes in amino acid content were found. Peptide mapping studies showed that a specific histidine was photooxidized. The sequence of the tetradecapeptide containing the photosensitive histidine is as follows: Val-Asp-Val-Val-Ala-Ile-Asn-Asp(Pro,Phe)Ile-Asp-Leu-His Photooxidation also inhibited oxidation of 3-phosphoglyceraldehyde by 50 to 60% suggesting that the histidine residue is involved in the principal physiological reaction carried out by the enzyme. At high dye to enzyme ratios, rose bengal in the dark also inhibited the esterase and dehydrogenase activities. Rose bengal was a competitive inhibitor with respect to DPN and arsenate, but a noncompetitive inhibitor with respect to 3-phosphoglyceraldehyde or p-nitrophenyl acetate. The effects of photooxidation produced a permanent change in the enzyme whereas the dark inhibitions were completely reversed by removal of the dye from the enzyme surface with charcoal treatment. The role of histidine in the various reactions catalyzed by the enzyme is discussed.