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Chemical and Stereochemical Actions of UDP-Galactose 4-Epimerase.
MedLine Citation:
PMID:  23339688     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Uridine(5')diphospho(1)α-d-galactose (UDP-gal) provides all galactosyl units in biologically synthesized carbohydrates. All healthy cells produce UDP-gal from uridine(5')diphospho(1)α-d-glucose (UDP-glc) by the action of UDP-galactose 4-epimerase (GalE). This Account provides our recent results describing unusual mechanistic features of this enzyme. Fully active GalE is dimeric and contains one tightly bound nicotinamide adenine dinucleotide (NAD) per subunit. The NAD undergoes reversible reduction to NADH in the chemical mechanism. GalE displays unusual enzymological, chemical, and stereochemical properties. These include practically irreversible binding of NAD, nonstereospecific hydride transfer, uridine nucleotide-induced activation of NAD, Tyr149 as a base catalyst, and [GalE-NADH]-oxidation in one-electron steps by one-electron acceptors. Early studies revealed that uridine(5')diphospho(1)α-d-4-ketopyranose (UDP-4-ketopyranose) and NADH are reaction intermediates. Weak binding of the 4-ketopyranosyl moiety and strong binding of the UDP-moiety allowed either face of the 4-ketopyranosyl moiety to accept hydride from NADH. In crystal structures of GalE, NAD bound within a Rossmann-type fold and uridine nucleotides within a substrate domain. Structures of [GalE-NADH] in complex with UDP-glc show Lys153, Tyr149, and Ser124 in contact with NAD or glucosyl-C4(OH). Lys153 forms hydrogen bonds to the ribosyl-OH groups of NAD. The phenolate of Tyr149 is associated with both the nicotinamide ring of NAD and glucosyl-C4(OH). Ser124 is hydrogen-bonded to glucosyl-C4(OH). Spectrophotometry studies show a pH-dependent charge transfer (CT) complex between Tyr149 and NAD. The CT-complex has a pK(a) of 6.1, which results in bleaching of the CT-band. The CT-band also bleaches upon binding of a uridine nucleotide. Kinetic experiments with wild-type GalE and Ser124Ala-GalE show the same kinetic pK(a) values as the corresponding CT-band pK(a), which point to Tyr149 as the base catalyst for hydride transfer. We used NMR studies to verify that uridine nucleotide binding polarizes nicotinamide π-electrons. The binding of uridine(5')-diphosphate (UDP) to GalE-[nicotinamide-1-(15)N]NAD shifts the (15)N-signal upfield 3 ppm, whereas UDP-binding to GalE-[nicotinamide-4-(13)C]NAD shifts the (13)C-signal downfield by 3.4 ppm. Electrochemical and (13)C NMR data for a series of N-alkylnicotinamides show that the 3.4 ppm downfield (13)C-perturbation in GalE corresponds to an elevation of the NAD reduction potential by 150 mV. These results account for the uridine nucleotide-dependence in the reduction of [GalE-NAD] by glucose or NaBH(3)CN. Kinetics in the reduction of Tyr149Phe- and Lys153Met-GalE-NAD implicate Tyr149 and Lys153 in the nucleotide-dependent reduction of NAD. They further implicate electrostatic repulsion between N1 of NAD and the ε-aminium group of Lys153 in nucleotide-induced activation of NAD. In an O(2)-dependent reaction, [GalE-NADH] reduces the stable radical UDP-TEMPO with production of superoxide radical. The reaction must proceed by way of a NAD-pyridinyl radical intermediate.
Authors:
Perry A Frey; Adrian D Hegeman
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-23
Journal Detail:
Title:  Accounts of chemical research     Volume:  -     ISSN:  1520-4898     ISO Abbreviation:  Acc. Chem. Res.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-23     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0157313     Medline TA:  Acc Chem Res     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Department of Biochemistry, University of Wisconsin-Madison , 1710 University Avenue, Madison, Wisconsin 53705, United States.
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