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Analysis of the galactosyltransferase reaction by positional isotope exchange and secondary deuterium isotope effects

Journal Article · · Arch. Biochem. Biophys.; (United States)
; ;
The mechanism of the galactosyltransferase-catalyzed reaction was probed using positional isotope exchange, alpha-secondary deuterium isotope effects, and inhibition studies with potential transition state analogs. Incubation of (beta-18O2, alpha beta-18O)UDP-galactose and alpha-lactalbumin with galactosyltransferase from bovine milk did not result in any positional isotope exchange. The addition of 4-deoxy-4-fluoroglucose as a dead-end inhibitor did not induce any detectable positional isotope exchange. alpha-Secondary deuterium isotope effects of 1.21 +/- 0.04 on Vmax and 1.05 +/- 0.04 on Vmax/KM were observed for (1-2H)-UDP-galactose. D-Glucono-1,5-lactone, D-galactono-1,4-lactone, D-galactono-1,5-lactone, nojirimycin, and deoxynojirimycin, did not inhibit the galactosyl transfer reaction at concentrations less than 1.0 mM. The magnitude of the secondary deuterium isotope effect supports a mechanism in which the anomeric carbon of the galactosyl moiety has substantial sp2 character in the transition state. Therefore, the cleavage of the bond between the galactose and UDP moieties in the transition state has proceeded to a much greater extent than the formation of the bond between the galactose and the incoming glucose. The lack of a positional isotope exchange reaction indicates that the beta-phosphoryl group of the UDP is not free to rotate in the absence of an acceptor substrate.
Research Organization:
Texas A and M Univ., College Station (USA)
OSTI ID:
6786655
Journal Information:
Arch. Biochem. Biophys.; (United States), Journal Name: Arch. Biochem. Biophys.; (United States) Vol. 267:1; ISSN ABBIA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
ANIMALS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BODY FLUIDS
CARBOHYDRATES
CATALYSIS
CATTLE
DEUTERIUM
DOMESTIC ANIMALS
ENERGY TRANSFER
ENZYMES
EVEN-EVEN NUCLEI
FOOD
GALACTOSE
GLUCOSE
HEXOSES
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPE EFFECTS
ISOTOPES
ISOTOPIC EXCHANGE
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MAMMALS
MATERIALS
MILK
MONOSACCHARIDES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXYGEN 18
OXYGEN ISOTOPES
REACTION KINETICS
RESONANCE
RUMINANTS
SACCHARIDES
STABLE ISOTOPES
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES