Identification and reactivity of the catalytic site of pig liver thioltransferase

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6030023

Gan, Z R; Wells, W W

The active site cysteine of pig liver thioltransferase was identified as Cys 22. The kinetics of the reaction between Cys 22 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)cystine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5 and no stable protein-cysteine disulfide was found suggesting an intramolecular disulfide formation. The reaction rate between reduced enzyme and S-sulfocysteine was concentration dependent, but not pH dependent, whereas the reaction between oxidized enzyme and reduced glutathione was both concentration and pH dependent. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys 22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys 22 and the substrate. Subsequently, the sulfhydryl group at Cys 25 is deprotonated as a result of microenvironmental 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 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: 6030023
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETIC ACID
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CYSTEINE
CYSTINE
DIGESTIVE SYSTEM
DISULFIDES
DOMESTIC ANIMALS
ENZYMES
GLANDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIVER
MAMMALS
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
REACTION KINETICS
SWINE
THIOLS
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES