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Predicate | Object |
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
48
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pubmed:dateCreated |
1994-1-6
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pubmed:abstractText |
UDP-galactose 4-epimerase contains NAD+ irreversibly but noncovalently bound to the active site. Uridine nucleotides bind to the substrate site and induce a protein conformational change that increases the chemical reactivity of NAD+ at the coenzyme site. Activation of NAD+ by uridine nucleotides perturbs the 15N and 13C NMR chemical shifts of selectively enriched NAD+ bound to the coenzyme site. The proton-decoupled 15N NMR signal for enzyme-bound [carboxamide-15N]NAD+ does not change upon addition of UDP, indicating that activation is not brought about by a change in the binding of the carboxamide group. The 15N NMR signal of enzyme-bound [nicotinamide-1-15N]NAD+ is shifted upfield 3.0 ppm and the 13C NMR signal for [nicotinamide-4-13C]NAD+ is shifted downfield 3.4 ppm downfield by the binding of UDP at the substrate site. These changes are consistent with the induction of a distortion into the nicotinamide ring, in which positive charge is transferred from N-1 to C-4. The kinetic and thermodynamic effects of these perturbations are significant, as indicated by the nonenzymatic chemical reactivities of a series of N-alkyl nicotinamides differing in the inductive electron withdrawing effects of the alkyl substituents. A downfield change of 3.4 ppm in the 4-13C chemical shifts brought about by electron withdrawal in the model compounds corresponds to a 3200-fold increase in the rate of reduction by NaBH3CN in water, a 15,000-fold increase in 86% ethanol, and a 152 mV more positive reduction potential in this series. The distortion of NAD+ by the binding of UDP is a long-range effect that is transmitted from the substrate binding site to the coenzyme through the protein conformational change. This apparently distorts the pi-electron distribution in the nicotinamide ring and reduces the activation energy for its reduction. Activation of enzyme-bound NAD+ toward reduction apparently arises from a destabilization in the nicotinamide ring structure rather than from a stabilization of the transition state through attractive interactions between the nicotinamide ring and the enzyme.
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pubmed:grant | |
pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Dec
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pubmed:issn |
0006-2960
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
7
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pubmed:volume |
32
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
13220-30
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:8241177-Bacterial Proteins,
pubmed-meshheading:8241177-Catalysis,
pubmed-meshheading:8241177-Escherichia coli,
pubmed-meshheading:8241177-Kinetics,
pubmed-meshheading:8241177-Magnetic Resonance Spectroscopy,
pubmed-meshheading:8241177-NAD,
pubmed-meshheading:8241177-Oxidation-Reduction,
pubmed-meshheading:8241177-Protein Binding,
pubmed-meshheading:8241177-Protein Conformation,
pubmed-meshheading:8241177-Structure-Activity Relationship,
pubmed-meshheading:8241177-Thermodynamics,
pubmed-meshheading:8241177-UDPglucose 4-Epimerase,
pubmed-meshheading:8241177-Uridine Diphosphate
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pubmed:year |
1993
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pubmed:articleTitle |
The importance of binding energy in catalysis of hydride transfer by UDP-galactose 4-epimerase: a 13C and 15N NMR and kinetic study.
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pubmed:affiliation |
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, U.S. Gov't, Non-P.H.S.,
Research Support, Non-U.S. Gov't
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