Contribution to Catalysis and Stability of the Five Cysteines in Escherichia Coli Aspartate Aminotransferase. Preparation and Properties of a Cysteine-Free Enzyme

Lisa M. Gloss, Jack F. Kirsch, Antoni Planas

Producción científica: Artículo en revista indizadaArtículorevisión exhaustiva

26 Citas (Scopus)

Resumen

The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A). Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S). A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed. The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation. The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry. The mutants had kat values up to 50% greater than that of WT and KAspM and Kα-KGM values up to 1.5- and 3.3-fold higher than that of WT. The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea. Quint is also less thermostable than WT, with a ΔTM of 3.3-4.4 °C. Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential. The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines. We consider the evolutionary implications of these findings.

Idioma originalInglés
Páginas (desde-hasta)32-39
Número de páginas8
PublicaciónBiochemistry
Volumen31
N.º1
DOI
EstadoPublicada - 1992

Huella

Profundice en los temas de investigación de 'Contribution to Catalysis and Stability of the Five Cysteines in Escherichia Coli Aspartate Aminotransferase. Preparation and Properties of a Cysteine-Free Enzyme'. En conjunto forman una huella única.

Citar esto