Inactivation of arginine esterase E-I of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone and isatoic anhydride

Int J Biochem. 1991;23(10):1101-10. doi: 10.1016/0020-711x(91)90150-l.

Abstract

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.

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Amino Acid Sequence
  • Arginine / analogs & derivatives
  • Arginine / pharmacology
  • Carboxylic Ester Hydrolases / antagonists & inhibitors*
  • Ethyldimethylaminopropyl Carbodiimide / pharmacology
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Molecular Structure
  • Oxazines / pharmacology
  • Solubility
  • Viper Venoms / enzymology*

Substances

  • Amino Acid Chloromethyl Ketones
  • Oxazines
  • Viper Venoms
  • Arginine
  • benzoylarginine ethyl ester
  • arginine esterase
  • Carboxylic Ester Hydrolases
  • phenylalanyl-prolyl-arginine-chloromethyl ketone
  • isatoic anhydride
  • Ethyldimethylaminopropyl Carbodiimide