tailieunhanh - Báo cáo khoa họcRe-engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild-type enzyme

Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2¢-phosphate of disfavoured NADPH, showed the expected large speci-ficity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofac-tors initially revealed little improvement with NADP + , although rates with NAD + were markedly diminished. | BFEBS Journal Re-engineering the discrimination between the oxidized coenzymes NAD and NADP in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild-type enzyme Marina Capone David Scanlonf Joanna Griffin and Paul C. Engel Schoolof Biomolecular and BiomedicalScience Conway Institute University College Dublin Ireland Keywords burst kinetics coenzyme purity coenzyme specificity glutamate dehydrogenase site-directed mutagenesis Correspondence P. C. Engel School of Biomolecular and Biomedical Science Conway Institute University College Dublin Belfield Dublin 4 Ireland Fax 353 1 716 6456 Tel 353 1 716 6764 E-mail Present address Kuros Biosurgery AG Zurich Switzerland fProgram in Neurosciences Mental Health Hospitalfor Sick Children Toronto Canada Received 5 March 2011 revised 21 April 2011 accepted 9 May 2011 doi Clostridial glutamate dehydrogenase mutants designed to accommodate the 2 -phosphate of disfavoured NADPH showed the expected large specificity shifts with NAD P H. Puzzlingly similar assays with oxidized cofactors initially revealed little improvement with NADP although rates with NAD were markedly diminished. This article reveals that the enzyme s discrimination in favour of NAD and against NADP had been greatly underestimated and has indeed been abated by a factor of 16 000 by the mutagenesis. Initially stopped-flow studies of the wild-type enzyme showed a burst increase of A340 with NADP but not NAD with amplitude depending on the concentration of the coenzyme rather than enzyme. Amplitude also varied with the commercial source of the NADP . FPLC HPLC and mass spectrometry identified NAD contamination ranging from to in different commercial samples. It is now clear that apparent rates of NADP utilization mainly reflected the reduction of contaminating NAD creating an entirely false view of the initial coenzyme specificity and also of the effects of

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