Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells.
Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata.
Swapna
S
Ghosh
Manju
M
Ray
Subhankar
S
Ray
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D020134:Catalytic Domain; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000096:biosynthesis* / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000737:chemistry; D007700:Kinetics; D051379:Mice
D019811:Hydroxylamine; D006639:Histidine; D004228:Dithionitrobenzoic Acid; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D004047:Diethyl Pyrocarbonate