Identification and reactivity of the catalytic site of pig liver thioltransferase
Journal Article
·
· J. Biol. Chem.; (United States)
OSTI ID:6409586
The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with (1-/sup 14/C)cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess (1-/sup 14/C)cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.
- Research Organization:
- Michigan State Univ., East Lansing
- OSTI ID:
- 6409586
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 14; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CYSTEINE
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
DRUGS
ENZYMES
GLANDS
GLUTATHIONE
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIVER
MAMMALS
MEMBRANE PROTEINS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PEPTIDES
POLYPEPTIDES
PROTEINS
RADIOPROTECTIVE SUBSTANCES
REACTION KINETICS
RECEPTORS
SWINE
THIOLS
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CYSTEINE
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
DRUGS
ENZYMES
GLANDS
GLUTATHIONE
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIVER
MAMMALS
MEMBRANE PROTEINS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PEPTIDES
POLYPEPTIDES
PROTEINS
RADIOPROTECTIVE SUBSTANCES
REACTION KINETICS
RECEPTORS
SWINE
THIOLS
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES