Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator
- Univ. of Wisconsin, Madison (USA)
UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5{prime}-diphosphate chloroacetol (UDC) and uridine 5{prime}-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K{sub D} of 0.110 mM and k{sub inact} of 0.84 min{sup {minus}1} at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD{sup +}. The inactivation of epimerase by uridine 5{prime}-diphosphate ({sup 2}H{sub 2})chloroacetol proceeds with a primary kinetic isotope effect (k{sub H}/k{sub D}) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD{sup +} at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD{sup +} is proposed to be the chromophore with {lambda}{sub max} at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.
- OSTI ID:
- 7118125
- Journal Information:
- Biochemistry; (USA), Vol. 29:9; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DEUTERIUM COMPOUNDS
ISOTOPE EFFECTS
ENOLS
BIOLOGICAL EFFECTS
ISOMERASES
INACTIVATION
ACETOLYSIS
BIOCHEMICAL REACTION KINETICS
ESCHERICHIA COLI
GALACTOSE
URIDINE
ALCOHOLS
ALDEHYDES
AZINES
BACTERIA
CARBOHYDRATES
CHEMICAL REACTIONS
DECOMPOSITION
ENZYMES
HETEROCYCLIC COMPOUNDS
HEXOSES
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
KINETICS
MICROORGANISMS
MONOSACCHARIDES
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PYRIMIDINES
REACTION KINETICS
RIBOSIDES
SACCHARIDES
SOLVOLYSIS
URACILS
550201* - Biochemistry- Tracer Techniques