title: 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.
abstract: 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 A(340) 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 0.04 to 0.37% 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 mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.
authors.affiliation: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland.
authors.forename: Marina
authors.initials: M
authors.lastname: Capone
authors.forename: David
authors.initials: D
authors.lastname: Scanlon
authors.forename: Joanna
authors.initials: J
authors.lastname: Griffin
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048009:Clostridium symbiosum / Q000201:enzymology* / Q000235:genetics; D004340:Drug Contamination; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000032:analysis / Q000378:metabolism*; D009249:NADP / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D007202:Indicators and Reagents; D050505:Mutant Proteins; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase