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Titolo:
Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase
Autore:
Drohat, AC; Jagadeesh, J; Ferguson, E; Stivers, JT;
Indirizzi:
Univ Maryland, Inst Biotechnol, Ctr Adv Res Biotechnol, Rockville, MD 20850 USA Univ Maryland Rockville MD USA 20850 Biotechnol, Rockville, MD 20850 USA Natl Inst Stand & Technol, Rockville, MD 20850 USA Natl Inst Stand & Technol Rockville MD USA 20850 Rockville, MD 20850 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 37, volume: 38, anno: 1999,
pagine: 11866 - 11875
SICI:
0006-2960(19990914)38:37<11866:ROEAGB>2.0.ZU;2-M
Fonte:
ISI
Lingua:
ENG
Soggetto:
CRITHIDIA-FASCICULATA; AMP NUCLEOSIDASE; STRUCTURAL BASIS; EXCISION-REPAIR; KINETICS; SPECIFICITY; HYDROLYSIS; ACID;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
31
Recensione:
Indirizzi per estratti:
Indirizzo: Stivers, JT Univ Maryland, Inst Biotechnol, Ctr Adv Res Biotechnol, 9600 Gudelsky Dr, Rockville, MD 20850 USA Univ Maryland 9600 Gudelsky Dr Rockville MD USA 20850 0850 USA
Citazione:
A.C. Drohat et al., "Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase", BIOCHEM, 38(37), 1999, pp. 11866-11875

Abstract

Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix [Stivers, J. T., et al. (1999) Biochemistry 38, 952]. A general acid-base mechanism has been proposed whereby His187 facilitates leaving group departure by protonating the O2 of uracil and Asp64 activates a water molecule for nucleophilic attack at Cl' of the deoxyribose. Detailed kinetic studies on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respectively, with little effect on substrate or product binding. The pH dependence of k(cat) for wild-type and H187Q UDG indicates that an unprotonated group in the enzyme-substrate complex (pK(a) = 6.2 +/- 0.2) is required for catalysis. This unprotonated group has a small Delta H of ionization (-0.4 +/- 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting that it corresponds to the general base Asp64. The pH dependence of k(cat) for wild-type, H187Q, and D64N UDG shows no evidence for an essential protonated group over the pH range of 5.5-10. Hence, the pK(a) of His187 must be outside this pH range if-it serves as an electrophilic catalyst. These results support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge on uracil O2 in the transition state. In the following paper of this issue we establish by crystallography and heteronuclear NMR spectroscopy that theimidazole of His 187 is neutral during the catalytic cycle of UDG.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/09/20 alle ore 17:17:21