Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification.
Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.
Ding
Shihua
S
Department of Fishery Science, Southwest Agricultural University, Chongqing 400716, PR China. shihuading@hotmail.com
Li
Yingwen
Y
Zhu
Liquan
L
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000663:Amphibians / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D006639:Histidine; D000469:Alkaline Phosphatase; D004047:Diethyl Pyrocarbonate