Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site
- State Univ. of New York, Stony Brook (USA)
Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl{sub 2}. 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 ({sup 3}H) 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.
- OSTI ID:
- 5471185
- Journal Information:
- Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 264:24; ISSN 0021-9258; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
560300* -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMIDES
AMINO ACIDS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
CARBONIC ACID DERIVATIVES
CARBOXYLIC ACIDS
CATALYSIS
CYSTEINE
ELEMENTS
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
FOOD
HYDROGEN COMPOUNDS
ISOMERASES
ISOTOPE APPLICATIONS
KINETICS
MEMBRANE PROTEINS
MERCURY
METALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PLANTS
PROTEINS
REACTION KINETICS
RECEPTORS
SOYBEANS
STOICHIOMETRY
THIOLS
THIOSULFATES
TIME DEPENDENCE
TRACER TECHNIQUES
TRITIUM COMPOUNDS
UREA
VEGETABLES