Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase.
3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites / Q000187:drug effects; D019759:Carbon-Nitrogen Lyases; D004047:Diethyl Pyrocarbonate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004793:Enzyme Reactivators; D005417:Flavobacterium / Q000201:enzymology; D005561:Formates / Q000494:pharmacology*; D006639:Histidine / Q000032:analysis*; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors*; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D004793:Enzyme Reactivators; D005561:Formates; D006639:Histidine; D008190:Lyases; D019759:Carbon-Nitrogen Lyases; C048147:3-ketovalidoxylamine A C-N lyase; D004047:Diethyl Pyrocarbonate