Histidine-254 is essential for the inactivation of phosphotriesterase with the alkynyl phosphate esters and diethyl pyrocarbonate.
The alkynyl phosphate ester, 1-hexynyl diethyl phosphate (I), is a mechanism-based inhibitor of phosphotriesterase. It has been previously determined that a histidine residue in the wild-type phosphotriesterase is covalently modified by this compound. In order to identify which of the seven histidine residues in the native enzyme are required for inactivation, the kinetic properties of phosphotriesterase mutants with this suicide substrate were examined in detail. Six of the seven mutants (histidine to asparagine) were rapidly inactivated by I. The mutants H55N, H57N, and H230N also showed partition ratios that were lower than for the wild-type enzyme. The rate of inactivation of H201N was significantly slower than that of wild-type phosphotriesterase. The H254N mutant could not be inactivated; no more than 60% of the initial activity was lost, even at I/E0 ratios of 4000:1. These results suggest that His-254 is essential for the inactivation of phosphotriesterase and is likely to be the primary target in the wild-type enzyme for modification by I. The inactivation of wild-type phosphotriesterase and the seven mutants was also studied using diethyl pyrocarbonate, a histidine selective reagent. The second-order rate constant for the inactivation of wild-type phosphotriesterase was determined to be 1.3 M-1 min-1. The rate constants for the inactivation of the H55N, H57N, H201N, and H230N mutants were larger than for the wild-type enzyme. Thus, it appears that when these histidine residues are replaced by asparagine, other histidine residues in the active site become more susceptible to modification, resulting in a faster rate of inactivation. The mutant H254N was not inactivated in the presence of DEPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Banzon
J A
JA
Department of Chemistry, Texas A&M University, College Station 77843.
Kuo
J M
JM
Fischer
D R
DR
Stang
P J
PJ
Raushel
F M
FM
D000480:Alkynes / Q000737:chemistry*; D043303:Aryldialkylphosphatase; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D004950:Esterases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry*; D011549:Pseudomonas / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D000480:Alkynes; D010710:Phosphates; D006639:Histidine; D004950:Esterases; D043303:Aryldialkylphosphatase; D004047:Diethyl Pyrocarbonate