@article {859,
	title = {Active site residues and mechanism of UDP-glucose dehydrogenase},
	journal = {European Journal of Biochemistry},
	volume = {271},
	number = {1},
	year = {2004},
	note = {ISI Document Delivery No.: 756CXTimes Cited: 16Cited Reference Count: 33},
	month = {Jan},
	pages = {14-22},
	type = {Article},
	abstract = {UDP-glucose dehydrogenase catalyzes the NAD(+)-dependent twofold oxidation of UDP-glucose to give UDP-glucuronic acid. A sequestered aldehyde intermediate is produced in the first oxidation step and a covalently bound thioester is produced in the second oxidation step. This work demonstrates that the Streptococcus pyogenes enzyme incorporates a single solvent-derived oxygen atom during catalysis and probably does not generate an imine intermediate. The reaction of UDP-[6",6"-di-H-2]-D-glucose is not accompanied by a primary kinetic isotope effect, indicating that hydride transfer is not rate determining in this reaction. Studies with a mutant of the key active site nucleophile, Cys260Ala, show that it is capable of both reducing the aldehyde intermediate, and oxidizing the hydrated form of the aldehyde intermediate but is incapable of oxidizing UDP-glucose to UDP-glucuronic acid. In the latter case, a ternary Cys260Ala/aldehyde intermediate/NADH complex is presumably formed, but it does not proceed to product as both release and hydration of the bound aldehyde occur slowly. A washout experiment demonstrates that the NADH in this ternary complex is not exchangeable with external NADH, indicating that dissociation only occurs after the addition of a nucleophile to the aldehyde carbonyl. Studies on Thr118Ala show that the value of k(cat) is reduced 160-fold by this mutation, and that the reaction of UDP-D-[6",6"-di-H-2]-glucose is now accompanied by a primary kinetic isotope effect. This indicates that the barriers for the hydride transfer steps have been selectively increased and supports a mechanism in which an ordered water molecule (H-bonded to Thr118) serves as the catalytic base in these steps.},
	keywords = {ACID, ALDEHYDE DEHYDROGENASE, CONSERVED CYSTEINE RESIDUES, DEHYDROGENASE, ENZYME, GDP-MANNOSE DEHYDROGENASE, GROUP-A STREPTOCOCCI, HISTIDINOL, MOLECULAR CHARACTERIZATION, PSEUDOMONAS-AERUGINOSA, URIDINE DIPHOSPHOGLUCOSE DEHYDROGENASE},
	isbn = {0014-2956},
	url = {<Go to ISI>://000187449200002},
	author = {Ge, X. and Penney, L. C. and van de Rijn, I. and Tanner, M. E.}
}
