Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site.
Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl2. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by [3H] N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
R A
RA
Bednar
W B
WB
Fried
Y W
YW
Lock
B
B
Pramanik
D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000378:metabolism; D019753:Intramolecular Lyases; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008626:Mercuribenzoates / Q000494:pharmacology; D008627:Mercuric Chloride / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology*; D010940:Plant Proteins / Q000378:metabolism*; D013776:Tetrathionic Acid / Q000494:pharmacology*; D013885:Thiosulfates / Q000494:pharmacology*; D014316:Tritium / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
C043407:4-mercuribenzoate; D008626:Mercuribenzoates; D010940:Plant Proteins; D013885:Thiosulfates; D014316:Tritium; D008627:Mercuric Chloride; D013776:Tetrathionic Acid; D014508:Urea; D007535:Isomerases; D019753:Intramolecular Lyases; C019865:chalcone isomerase; D008628:Mercury; D003545:Cysteine; D005033:Ethylmaleimide