Inactivation of arginine esterase E-I of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone and isatoic anhydride.
1. Esterase E-I from Bitis gabonica was inactivated with irreversible inhibitors which included studies with a water-soluble carbodiimide, an affinity labelling peptide and a mechanism-based inactivator. 2. The reaction with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide was biphasic and the dominant part followed saturation kinetics. At pH 5.5 a rate constant of 0.4 min-1 for inactive enzyme formation was calculated and a dissociation constant (Ki) of 0.2 M for the enzyme-inhibitor complex. 3. Inactivation with D-Phe-Pro-Arg-chloromethyl ketone indicated a two-step mechanism, for which the reaction parameters at pH 8.0 were determined. The Ki value was 0.2 microM and the inactivation rate was 2.5 min-1. 4. With isatoic anhydride pseudo-first-order kinetics was observed. At pH 8.0 a rate constant of 0.9 min-1 and a Ki of 2.0 mM were obtained. The inactivation of the enzyme was found to be governed by a group in the enzyme showing a pK value of 7.3.
Gravett
P S
PS
Health Chemical Laboratory, Department of National Health and Population Development, Pretoria, Republic of South Africa.
Viljoen
C C
CC
Oosthuizen
M M
MM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010078:Oxazines / Q000494:pharmacology; D012995:Solubility; D014757:Viper Venoms / Q000201:enzymology*
D000590:Amino Acid Chloromethyl Ketones; D010078:Oxazines; D014757:Viper Venoms; D001120:Arginine; C009727:benzoylarginine ethyl ester; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone; C037902:isatoic anhydride; D005022:Ethyldimethylaminopropyl Carbodiimide