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Titolo:
tRNA-guanine transglycosylase from Escherichia coli: Molecular mechanism and role of aspartate 89
Autore:
Kittendorf, JD; Barcomb, LM; Nonekowski, ST; Garcia, GA;
Indirizzi:
Univ Michigan, Coll Pharm, Dept Med Chem, Ann Arbor, MI 48109 USA Univ Michigan Ann Arbor MI USA 48109 pt Med Chem, Ann Arbor, MI 48109 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 47, volume: 40, anno: 2001,
pagine: 14123 - 14133
SICI:
0006-2960(20011127)40:47<14123:TTFECM>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
ACTIVE-SITE NUCLEOPHILE; TRANSFER-RNA; CATALYTIC NUCLEOPHILE; AMP NUCLEOSIDASE; DIRECTED MUTAGENESIS; ZYMOMONAS-MOBILIS; RECOGNITION; IDENTIFICATION; 2-DEOXYRIBOSYLTRANSFERASE; PARTICIPATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
43
Recensione:
Indirizzi per estratti:
Indirizzo: Garcia, GA Univ Michigan, Coll Pharm, Dept Med Chem, Ann Arbor, MI 48109 USA Univ Michigan Ann Arbor MI USA 48109 , Ann Arbor, MI 48109 USA
Citazione:
J.D. Kittendorf et al., "tRNA-guanine transglycosylase from Escherichia coli: Molecular mechanism and role of aspartate 89", BIOCHEM, 40(47), 2001, pp. 14123-14133

Abstract

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a posttranscriptional transglycosylation reaction involved in the incorporationof the modified base queuine [Q, 7-(4,5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl)-7-deazaguanine] into tRNA. Previously, the crystal structure of the TGT from Zymomonas mobilis was solved in complex with preQ(1) (the substrate for the eubacterial TGT) [Romier et al. (1996) EMBO J. 15, 2850-2857]. An aspartate residue at position 102 (position 89 in the Escherichia coliTGT) was proposed to play a nucleophilic role in an associative catalytic mechanism. Although this is an attractive and precedented mechanism, a dissociative mechanism is equally plausible. In a dissociative mechanism, aspartate 89 would provide electrostatic stabilization of an oxocarbenium ion intermediate that is formed by dissociation of guanine. To clarify the natureof the catalytic mechanism of TGT, we have generated and characterized four mutations of aspartate 89 in the E. coli TGT (alanine, asparagine, cysteine, and glutamate). All four mutant TGTs were able to noncovalently bind tRNA, but only the glutamate mutant was able to form a stable complex with the RNA substrate under denaturing conditions that was comparable to wild type. Furthermore, the glutamate mutant was the only mutant TGT that demonstrated significant activity. Kinetic parameters were determined for this enzyme and shown to be comparable to wild type, revealing that the enzyme is considerably tolerant of the positioning of the carboxylate. Under conditions of high enzyme concentrations and long time courses, the alanine, asparagine, and cysteine mutants showed very low levels (ca. 10(3)-fold lower than wild type) of activity that were linear with respect to enzyme concentrationand dependent upon pH in a fashion similar to that of the wild type. However, the observed initial velocities were too low to accurately determine k(cat) and K-m values. We hypothesize that the activity observed for these mutants is most likely derived from host strain TGT (wt) contamination. Theseresults are most consistent with aspartate 89 acting as a nucleophile in an associative catalytic mechanism.

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Documento generato il 18/09/20 alle ore 08:52:10