tailieunhanh - Báo cáo khoa học: Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis

Glutamate dehydrogenase (EC –4) fromPeptostreptococcus asacch-arolyticushas a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms ofkcat ⁄Kmvalues. Sequence alignments across the wider family of NAD(P)-dependent dehydrogenases might suggest that this preference is mainly due to a negatively charged glutamate at position 243 (E243) in the adenine ribose-binding pocket. | ỊFEBS Journal Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis John B. Carrigan and Paul C. Engel Schoolof Biomolecular and BiomedicalScience Conway Institute University College Dublin Ireland Keywords coenzyme specificity glutamate dehydrogenase NAD P nicotinamide nucleotides site-directed mutagenesis Correspondence P. C. Engel Schoolof Biomolecular and BiomedicalScience Conway Institute University College Dublin Belfield Dublin 4 Ireland Fax 353 1283 7211 Tel 353 1716 6764 E-mail Received 31 May 2007 revised 10 July 2007 accepted 10 August 2007 doi Glutamate dehydrogenase EC from Peptostreptococcus asacch-arolyticus has a strong preference for NADH over NADPH as a coenzyme over 1000-fold in terms of kcat Km values. Sequence alignments across the wider family of NAD P -dependent dehydrogenases might suggest that this preference is mainly due to a negatively charged glutamate at position 243 E243 in the adenine ribose-binding pocket. We have examined the possibility of altering coenzyme specificity of the Peptostreptococcus enzyme and more specifically the role of residue 243 and neighbouring residues in coenzyme binding by introducing a range of point mutations. Glutamate dehydrogenases are unusual among dehydrogenases in that NADPH-spe-cific forms usually have aspartate at this position. However replacement of E243 with aspartate led to only a nine-fold relaxation of the strong discrimination against NADPH. By contrast replacement with a more positively charged lysine or arginine as found in NADPH-dependent members of other dehydrogenase families allows a more than 1000-fold shift toward NADPH resulting in enzymes equally efficient with NADH or NADPH. Smaller shifts in the same direction were also observed in enzymes where a neighboring tryptophan W244 was replaced by a smaller alanine approximately six-fold or .

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