Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase.
The reactivity of simple alkyl thiolates with N-ethylmaleimide (NEM) follows the Brønsted equation, log kS- = log G + beta pK, with G = 790 M-1 min-1 and beta = 0.43. The rate constant for the reaction of the thiolate of 2-mercaptoethanol with NEM is 10(7) M-1 min-1, whereas the rate constant for the reaction of the protonated thiol is less than 0.0002 M-1 min-1. The intrinsic reactivity of the protonated thiol (SH) is over (5 X 10(10]-fold less than the thiolate (S-) and makes a negligible contribution to the reactivity of thiols toward NEM. The rate of NEM modification of chalcone isomerase was conveniently measured by following the concomitant loss in enzymatic activity. The pseudo-first-order rate constants for inactivation show a linear dependence on the concentration of NEM up to 200 mM and yield no evidence for noncovalent binding of NEM to the enzyme. Evidence is presented demonstrating that the modification of chalcone isomerase by NEM is limited to a single cysteine residue over a wide range of pH. Kinetic protection against inactivation and modification by NEM is provided by competitive inhibitors and supports the assignment of this cysteine residue to be at or near the active site of chalcone isomerase. The pH dependence of inactivation of the enzyme by NEM indicates a pK of 9.2 for the cysteine residue in chalcone isomerase. At high pH, the enzymatic thiolate is only (3 X 10(-5))-fold as reactive as a low molecular weight alkyl thiolate of the same pK, suggesting a large steric inhibition of reaction on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Bednar
R A
RA
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019753:Intramolecular Lyases; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D013025:Glycine max / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D007535:Isomerases; D019753:Intramolecular Lyases; C019865:chalcone isomerase; D005033:Ethylmaleimide