Insights into the reaction mechanism of the diisopropyl fluorophosphatase from Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis.
Kinetic measurements, chemical modification and site-directed mutagenesis have been employed to gain deeper insights into the reaction mechanism of the diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. Analysis of the kinetics of diisopropyl fluorophosphate hydrolysis reveals optimal enzyme activity at pH >/=8, 35 degrees C and an ionic strength of 500 mM NaCl, where k(cat) reaches a limiting value of 526 s(-1). The pH rate profile shows that full catalytic activity requires the deprotonation of an ionizable group with an apparent pK(a) of 6.82, DeltaH(ion) of 42 kJ/mol and DeltaS(ion) of 9.8 J/mol K at 25 degrees C. Chemical modification of aspartate, glutamate, cysteine, arginine, lysine and tyrosine residues indicates that these amino acids are not critical for catalysis. None of the six histidine residues present in DFPase reacts with diethyl pyrocarbonate (DEPC), suggesting that DEPC has no accessibility to the histidines. Therefore, all six histidine residues have been individually replaced by asparagine in order to identify residues participating in catalysis. Only substitution of H287 renders the enzyme catalytically almost inactive with a residual activity of approx. 4% compared to wild-type DFPase. The other histidine residues do not significantly influence the enzymatic activity, but H181 and H274 seem to have a stabilizing function. These results are indicative of a catalytic mechanism in which H287 acts as a general base catalyst activating a nucleophilic water molecule by the abstraction of a proton.
Hartleib
J
J
Institute of Biophysical Chemistry, Johann Wolfgang Goethe University of Frankfurt/M., Marie-Curie-Strasse 9, 60439, Frankfurt/M., Germany.
Rüterjans
H
H
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002384:Catalysis / Q000187:drug effects; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D049832:Decapodiformes / Q000201:enzymology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D007531:Isoflurophate / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D044345:Phosphoric Triester Hydrolases; D013696:Temperature; D013816:Thermodynamics; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology
D002800:Cholinesterase Inhibitors; D007093:Imidazoles; D007531:Isoflurophate; D006639:Histidine; D014302:Trinitrobenzenesulfonic Acid; D004228:Dithionitrobenzoic Acid; D004950:Esterases; D044345:Phosphoric Triester Hydrolases; C020061:diisopropyl-fluorophosphatase; D005022:Ethyldimethylaminopropyl Carbodiimide; C012160:N-acetylimidazole