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 Scanlon, Joanna Griffin and Paul C. Engel School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland Introduction The nicotinamide-nucleotide-dependent dehydrogenases tend, in general, to be either NAD + -specific (and then catabolic) or NADP(H)-specific (and accordingly ana- bolic, except for those few enzymes such as glucose 6-phosphate dehydrogenase which provide NADPH for biosynthesis) [1]. Crystallographic studies of arche- typal NAD + -specific enzymes, such as alcohol and lactate dehydrogenases [2,3], and archetypal NADPH- specific dehydrogenases such as glutathione reductase [4] have offered some degree of understanding of the ways in which these enzymes achieve their coenzyme specificity. This has been augmented by various detailed studies of amino acid sequences [5,6], and has been both tested and applied in some notably success- ful examples of re-engineering of coenzyme specificity [7–19]. As noted by Khouri et al. [17], however, the 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: paul.engel@ucd.ie Present address *Kuros Biosurgery AG, Zu ¨ rich, Switzerland Program in Neurosciences & Mental Health, Hospital for Sick Children, Toronto, Canada (Received 5 March 2011, revised 21 April 2011, accepted 9 May 2011) doi:10.1111/j.1742-4658.2011.08172.x 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. 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 con- taminating 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 esti- mated 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 struc- tural 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. 2460 FEBS Journal 278 (2011) 2460–2468 ª 2011 The Authors Journal compilation ª 2011 FEBS lessons learned from previous attempts to modify coenzyme specificity cannot be safely generalized to other systems. The terms NAD + - or NADPH-specific seem to imply absolute discrimination between the closely simi- lar coenzymes, but discrimination is never total, and the actual factor varies widely from enzyme to enzyme. Particularly interesting, however, are those enzyme families that include members showing little discrimination, so-called dual-specificity dehydrogenas- es. The glutamate dehydrogenases are such a family [20] and, as a result, have three different EC classifica- tions, EC 1.4.1.2, EC 1.4.1.3 and EC 1.4.1.4 for NAD + -specific, dual-specific (particularly common in higher animals and in Archaea), and NADP + -specific members, respectively. Most of these are evolutionarily and structurally related, in many cases quite closely, despite the functional and classificational division [21,22], and thus they provide a revealing example of the way in which a single structural scaffold can be adapted to produce remarkably different functional outcomes. Our own protein engineering experiments [23], based on an analysis of the high-resolution structure of the binary enzyme–NAD + complex of clostridial gluta- mate dehydrogenase [24,25], were aimed initially at facilitating productive binding of the phosphorylated coenzyme by enlarging the potential binding pocket and removing the negative charge, likely to repel the 2¢-phosphate of NADP(H), and replacing it with posi- tive charge. Accordingly, mutants F238S, P262S and F238S ⁄ P262S were created to provide more space, and D263K to offer a more favourable electrostatic envi- ronment. The kinetic behaviour of these mutant enzymes with NADH and NADPH was compared at different pH values [23], and, especially at the highest pH examined (8), there were large changes in the dis- crimination factor, so that, although none of the mutants showed a complete reversal of specificity, the last two of the four listed could reasonably be described as dual-specific. When, however, we turned to the opposite direction of reaction, there was a perplexing difference in the results, with seemingly remarkably little change in the strong preference for NAD + over NADP + , estab- lished as 300-fold under standard assay conditions by Syed et al. [26]. In this article, we document these surprising results and then analyse the source of the discrepancy, with an outcome that not only necessi- tates a revised view of clostridial glutamate dehydro- genase and its specificity, but also has wider significance for the study of coenzyme specificity in other enzymes. Results and Discussion Initial kinetic analysis The coenzyme specificity of the mutant enzymes was initially assessed using the best available commercial coenzymes without further purification. Values of k cat , K m and k cat ⁄ K m for the oxidized coenzymes are pre- sented in Table 1. After replacement of Phe238 by serine, NAD + was less effective as a coenzyme because of moderate decreases in k cat (36, 14 and 33% at pH 6.0, 7.0 and 8.0, respectively) and marked increases in apparent K m ( 10 fold at pH 6.0 and 7.0, 14 fold at pH 8.0). NAD + is evidently bound very poorly to this mutant. However, surprisingly, no improvement was apparent with NADP + as the coenzyme. At pH 7.0 and 8.0, approximately five-fold decreases in k cat , and increases of approximately four- and six-fold respec- tively for K m (Table 1), appeared to indicate markedly lower overall catalytic efficiency with this coenzyme. The single proline to serine mutation at position 262 likewise decreased the overall catalytic efficiency with both oxidized coenzymes. Using NAD + , this mutant had values comparable with wild-type for k cat , particu- larly at pH 7.0 and 8.0 (Table 1). Decreases in cata- lytic efficiency were due to increases in K m , approximately nine-fold at pH 7.0 and seven-fold at pH 8.0. For NADP + , the decrease in overall catalytic efficiency reflected decreases in k cat of almost three-fold at pH 8.0, with accompanying increases in K m , approx- imately five-fold at pH 7.0 and three-fold at pH 8.0 (Table 1). Correspondingly, this mutation seemed to offer no significant shift in specificity towards NADP + . Turning to the third of the single mutants, D263K, once again, there appeared to be a decrease in catalytic efficiency with both NAD + and NADP + as coen- zymes, though less marked than for the other muta- tions, and at pH 8.0 with NADP + as coenzyme there was little difference between the performance of the wild-type and mutant enzymes (Table 1). The results for these three mutants were extremely puzzling; the mutations had been designed to facilitate binding of the phosphorylated coenzyme, and with the reduced coenzymes [23] there were indeed large shifts, as expected, in the discrimination factor, 150 fold and 272 fold, respectively, for example, for P262S and D263K at pH 8.0. In this study, only the double- mutant F238S ⁄ P262S gave a result reasonably close to expectation with the oxidized coenzymes (Table 1): at both pH 7.0 and pH 8.0 the large discrimination factor in favour of NAD + decreased to only 3–4 for this mutant. Even in this case, however, the apparent M. Capone et al. Coenzyme preference in glutamate dehydrogenase FEBS Journal 278 (2011) 2460–2468 ª 2011 The Authors Journal compilation ª 2011 FEBS 2461 improvement was entirely due to a large decrease in catalytic efficiency with NAD + rather than an increase with NADP + . In fact, there was a deterioration of  10 fold in the catalytic efficiency with NADP + . Thus here also, the results were in contrast to those for the reduced coenzymes [23], which, at pH 7.0 and 8.0, showed large increases in catalytic efficiency with NADPH, over 100-fold at pH 8.0. Rapid-reaction studies The possibility that different rate-limiting steps in the two reaction directions might account for the strikingly different outcomes of the mutagenesis with reduced [23] and oxidized coenzymes (above) prompted an investigation of presteady-state kinetics. Burst kinetics detects rapid accumulation of product before the steady state is reached: the presence or absence of a burst provides information on the position of the rate- limiting step along the reaction pathway, and the amplitude of the burst should be proportional to the enzyme concentration. A ‘burst’ increase in A 340 was detected in the first few milliseconds of reaction with NADP + as coenzyme, but not with NAD + . Two dif- ferent phases were identified in the stopped-flow traces: the first phase (Fig. 1A inset) consisted of the rapid single exponential burst in A 340 , reaching an apparent plateau within a few seconds. The small differences in the height of this plateau for different concentrations of enzyme (5–20 lm) are due to shifts in the baseline as a result of the contribution of the enzyme itself to A 340 ; after correction for this baseline shift (Fig. 1B), the burst amplitude was entirely independent of the enzyme concentration. Over much longer periods (Fig. 1A, main panel), the apparent plateau was revealed as a very slow and initially linear second phase of increase in absorbance, finally leading to the expected reaction equilibrium in over 4 h. Further analysis showed that the burst amplitude with NADP + was dependent on the concentration of the coenzyme itself, and not only on its concentration, but also the commercial source. With 1 mm NADP + from Roche Diagnostics Ltd. (Burgess Hill, UK), the burst amplitude corresponded to 3.1 lm reduced coenzyme, i.e. 0.31% of the NADP + used. Similarly, a burst of 1.2 lm newly formed reduced coenzyme, was observed for NADP + from Apollo Scientific (Stockport, UK), corresponding to 0.12% of the total NADP + . Analysis and purification of NADP + The rapid-reaction results strongly suggested that the burst might be due to trace impurities in the coenzyme. Direct HPLC analysis of the same NADP + batches (Fig. 2) revealed trace contamination. Despite the com- plexity of the chromatograms, the injection of NADP + Table 1. Initial comparison of kinetic parameters between wild-type glutamate dehydrogenase, F238S, P262S, F238S ⁄ P262S and D263K mutant enzymes. To determine kinetic parameters for NAD ⁄ P + , glutamate concentration was kept constant (40 mM) over a range of NAD ⁄ P + concentrations (0.01–1 mM) under standard assay conditions. All experiments were repeated in triplicate and the kinetic parameters and their standard errors (± SE) were calculated by a nonlinear regression method [36] with ENZPACK version 3.0 (Biosoft Ltd, Cambridge, UK). The discrimination factor in the right-hand column, a measure of the preference for NAD + over NADP + , is calculated as the ratio of the catalytic efficiency, k cat ⁄ K m , for NAD + to that for NADP + . ND, not determined. pH NAD + NADP + Discrimination factor k cat (s )1 ) K m (mM) k cat ⁄ K m (s )1 ÆmM )1 ) k cat (s )1 ) K m (mM) k cat ⁄ K m (s )1 ÆmM )1 ) Wild-type 6.0 3.88 ± 0.09 0.10 ± 0.02 38.8 0.13 ± 0.05 0.58 ± 0.01 0.224 173 F238S 6.0 1.41 ± 0.02 1.00 ± 0.05 1.41  0.07 ND ND P262S 6.0 0.51 ± 0.02 0.314 ± 0.034 1.63 ND ND F238S ⁄ P262S 6.0 ND ND ND D263K 6.0 1.92 ± 0.07 0.18 ± 0.02 10.7  0.09 ND ND Wild-type 7.0 20.4 ± 0.7 0.114 ± 0.012 179 0.57 ± 0.06 0.26 ± 0.01 2.19 81.7 F238S 7.0 17.6 ± 0.3 1.25 ± 0.46 14.1 0.125 ± 0.04 1.1 ± 0.7 0.113 125 P262S 7.0 23.0 ± 1.4 1.04 ± 0.13 23.0 0.457 ± 0.05 1.23 ± 0.22 0.371 62 F238S ⁄ P262S 7.0 3.31 ± 0.47 2.22 ± 0.21 1.49 0.242 ± 0.05 0.632 ± 0.27 0.382 3.9 D263K 7.0 12.6 ± 0.21 0.17 ± 0.02 72.4 0.40 ± 0.05 0.380 ± 0.002 1.05 69 Wild-type 8.0 51.6 ± 0.6 0.127 ± 0 .012 482 0.909 ± 0.19 0.336 ± 0.015 2.70 179 F238S 8.0 39.8 ± 1.7 1.83 ± 0.04 21.7 0.270 ± 0.05 2.04 ± 0.08 0.132 164 P262S 8.0 53.4 ± 2.6 0.90 ± .11 59.3 0.339 ± 0.06 1.04 ± 0.30 0.339 175 F238S ⁄ P262S 8.0 4.49 ± 0.34 6.28 ± 0.34 0.71 0.513 ± 0.193 2.24 ± 0.77 0.229 3.1 D263K 8.0 39.3 ± 1.9 0.376 ± 0.03 104 0.958 ± 0.05 0.336 ± 0.05 2.85 36.5 Coenzyme preference in glutamate dehydrogenase M. Capone et al. 2462 FEBS Journal 278 (2011) 2460–2468 ª 2011 The Authors Journal compilation ª 2011 FEBS enriched with NAD + permitted identification of a peak of the latter estimated at  0.37% in NADP + from Roche,  0.15% in NADP + from Apollo Scientific and  0.04% in NADP + from Sigma-Aldrich Ireland Ltd. (Dublin, Ireland). The agreement of the rapid reaction kinetics with the HPLC analysis suggested nearly total conversion of NAD + into NADH in the reaction observed. From the equilibrium constant for the oxida- tive deamination of l-glutamate [27] it can be estimated that, under the conditions used,  97–98% of the con- taminant NAD + should be converted into NADH. These results strongly suggest that NAD + is the con- taminant affecting the course of the enzymatic assays. Stopped-flow experiment repeated with purified NADP + When the rapid-reaction experiment was repeated under identical conditions but using NADP + from Apollo Scientific freshly purified in our laboratory, the absence of the ‘burst’ (Fig. 3) confirmed that this phe- nomenon was due to the impurities in the commercial coenzyme. In addition, when commercial NADP + was used without purification for the steady-state kinetics, an inhibitory effect of the NADH formed in the first part of the reaction was observed on the following phase of the reaction with NADP + . This effect was confirmed by enriching the mixture with varying amounts of NADH (results not shown). The binding of the reduced coenzymes to the active site of clostrid- ial glutamate dehydrogenase is much tighter than the binding of the oxidized coenzyme. Together with the initial preference for the nonphosphorylated coenzyme, this explains the potent inhibitory effect of such a small NADH contamination. Mass spectrometric identification of NAD + peak Isolation and mass spectroscopic analysis confirmed the identity of the contaminant. Comparison with the spectrum of an authentic NAD + sample revealed total similarity of the fragmentation pattern. The neg- ative portion of the spectrum displayed a fragment at m ⁄ z 540, along with a small amount of parental mol- ecule (m ⁄ z 662.1). The signal at m ⁄ z 540 corresponds to the ADP-ribose moiety of the coenzyme resulting from splitting off the nicotinamide ring, suggesting that the covalent bond between the nicotinamide and the ribose of the coenzyme is particularly labile. The signal of the parental molecule is also visible at m ⁄ z 664.1 in the positive spectrum; the nicotinamide ring, counterpart fragment of the ADP-ribose (m ⁄ z 540) gives a signal at m ⁄ z 123.1, whereas ade- nine is registered at m ⁄ z 136.1. In view of these findings, the possibility of the reverse contamination was also tested. However, the 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 150 200 250 300 A 340 nm A 340 nm Time (min) 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 [enzyme] (μM) A B Fig. 1. Time course of the reaction of wild-type clostridial gluta- mate dehydrogenase with NADP + . (A) The main panel shows the reaction of 15 l M glutamate dehydrogenase with 1 mM coenzyme and 40 m ML-glutamate (concentrations after mixing) monitored at 340 nm over 250 min. The increase in absorbance was linear for the first 30–40 min; on this basis, the value for the specific activity was calculated as 1.84 nmolÆmin )1 Æmg )1 , rate 0.0015 s )1 . The inset shows stopped-flow traces observed over the first few seconds of the forward reaction of 2.5 l M (lowest trace), 5 lM, 7.5 lM, 10 lM and 15 lM (highest trace) wild-type clostridial glutamate dehydroge- nase with 1 m M NADP + and 40 mML-glutamate (all concentrations after mixing). A burst phase was detected in all cases. The almost horizontal trace seen in each case after 2–3 s corresponds to the slow, steady-state reaction monitored in the main panel. (B) Cor- rection applied to the burst amplitudes calculated at different enzyme concentrations in (A). The increase of enzyme concentra- tion causes a significant baseline shift at 340 nm. A reference zero baseline was first recorded by mixing 2 m M NADP + and 80 mML- glutamate with buffer in the stopped-flow; a set of individual base- lines was recorded by mixing different enzyme concentrations with buffer (d); finally, 2 m M NADP + and 80 mML-glutamate were mixed with different enzyme concentrations, and the burst ampli- tudes were recorded (s). The baseline recorded for each enzyme concentration (d) was subtracted from the corresponding value recorded for the burst amplitude (s), giving the final plot (.) showing no dependence of the burst amplitude on the enzyme concentration. M. Capone et al. Coenzyme preference in glutamate dehydrogenase FEBS Journal 278 (2011) 2460–2468 ª 2011 The Authors Journal compilation ª 2011 FEBS 2463 contamination of NAD + by NADP + reported by some other authors [28] was not observed in our batches of NAD + . Reassessment of coenzyme specificity of clostridial glutamate dehydrogenase In light of the discovery of variable contamination of the commercial NADP + samples by the preferred coen- zyme NAD + , it was necessary to reconsider the steady- state results. First of all, the specific activity of clostrid- ial glutamate dehydrogenase under standard assay con- ditions (1 mm coenzyme, 40 mml-glutamate, 0.1 m phosphate, pH 7.0) was re-determined with freshly purified NADP + and using three different methods of measurement, absorbance measurements in the stopped- flow apparatus, conventional spectrophotometry and fluorimetry. These three methods, respectively, yielded values of 2.22, 2.33 and 2.78 · 10 )3 lmolÆmin )1 Æmg )1 (mean = 2.44 · 10 )3 lmolÆmin )1 Æmg )1 ). This figure is  11 000 times lower than the corresponding figure for the preferred coenzyme NAD + . This remarkable dis- crimination factor is nearly 40 times higher than the 300-fold factor reported by Syed et al. [26]. It is now also very clear how such a large overestimation of the rate with NADP + can arise. If we assume the use of a commercial NADP + containing 0.3% NAD + , as in the case of the Roche sample used in this study, then in a steady-state assay, as in the rapid-reaction study, the contaminating NAD + will be used first. There will not be simple proportionality because the 1 mm NAD + in a standard assay is well above K m and the 0.3% NAD + contamination in 1 mm NADP + is far below K m . Nevertheless, a rate  1 ⁄ 200 of the rate in a standard NAD + reaction may be anticipated. A detailed re-analysis of the steady-state properties of wild-type glutamate dehydrogenase with freshly purified NADP + was therefore carried out. The values for K m and k cat given in Table 1 are 0.26 ± 0.01 mm and 0.57 ± 0.06 s )1 , respectively. The redetermination with pure coenzyme gave a much higher value for the K m of 3.2 ± 0.4 mm, 30-fold higher than the K m for NAD + . Moreover, k cat was calculated as 8.2 · 10 )3 ±6· 10 )4 s )1 ,  2500 fold lower than the figure for NAD + and also 70 times lower than the value obtained with unpurified coenzyme. On this basis, a new ratio of the specificity constants for the oxidized coenzymes can be calculated: this ‘discrimina- tion factor’ reveals that wild-type clostridial glutamate dehydrogenase is  80 000-fold more active with NAD + than with NADP + . This factor was previously estimated as 82 at pH 7.0 (Table 1), 1000 times less Time (min) a.u. Fig. 2. Overlap of HPLC elution profiles of NADP + batches from different suppliers. The four traces show the impurity peaks in 25–30 ng samples of commercial NADP + (black, Roche; green, Apollo Scientific; blue, Sigma; cyan, Apollo enriched with NAD + ). The last sample allowed unambiguous iden- tification of the peak of NAD + in the other chromatograms (indicated by the arrow). The amounts of contaminant NAD + present in the samples were calculated by peak integration using the MILLENNIUM software package. Fig. 3. Superposition of stopped-flow burst-kinetic traces for wild- type clostridial glutamate dehydrogenase and different batches of NADP + at identical concentrations. (Upper) Reaction with Grade I NADP + from Roche. (Middle) Reaction with NADP + from Apollo Scientific. (Lower) Reaction with freshly purified NADP + . Coenzyme preference in glutamate dehydrogenase M. Capone et al. 2464 FEBS Journal 278 (2011) 2460–2468 ª 2011 The Authors Journal compilation ª 2011 FEBS than the true value, underlining the impact of quite a small level of contamination on these results. Reassessment of the effects of the mutations on coenzyme specificity In view of the dramatically altered figure for the strin- gency of coenzyme specificity in clostridial glutamate dehydrogenase, it now also becomes clear that the ini- tial assessment of the effects of the mutagenesis on the relative activities with NAD + and NADP + is very likely to be misleading. As a preliminary test of this, kinetic parameters were redetermined using freshly purified NADP + at pH 7.0 for two mutants, F238S which had appeared to give deterioration rather than improvement with NADP + , and the double-mutant which showed a 21-fold improvement from a discrimi- nation ratio of 82 to one of 3.9. The data in Table 2 show a remarkable change in this assessment. For the wild-type enzyme with NADP + , the true values for k cat are very much lower and those for K m much higher than previously estimated with the commercial coenzyme. As a result, the discrimination factor for the wild-type enzyme is underestimated by  1000- fold. F238S, therefore, offers a 161-fold improvement instead of a 1.5-fold deterioration in discrimination factor. Even more dramatically, the modest apparent 21-fold improvement in the double-mutant should be a 16 200-fold improvement, entirely vindicating the ini- tial thinking behind the mutagenesis. Wider implications Careful purification of nicotinamide coenzymes has been recognized in the past as an important issue in the study of dehydrogenases [29]: coenzymes were often purified in research laboratories prior to use [30,31] in order to avoid misleading kinetic anomalies, but this routine has largely been abandoned in recent years because the purity and stability of the best commercial preparations have dramatically improved. Although concern and worry persist over the purity of reduced coenzymes, which are often contaminated by the oxidized form, Grade I NAD + and NADP + are generally utilized without further purification, even in enzymatic studies of coenzyme specificity [7,32,33]. In our own laboratory, because analytical HPLC only revealed what we took to be trace, negligible contami- nation, well below 0.5%, we have frequently proceeded without further purification of the coenzyme. Other authors, using sensitive detection with a dehydrogenase coupled with the reduction of INT to a coloured for- mazan, have recently reported the presence of  0.1% NAD + in NADP + from different suppliers [34,35]. Woodyer et al. [28] also mentioned coenzyme contami- nation in the context of coenzyme specificity studies of the NAD + -dependent phosphite dehydrogenase from Pseudomonas stutzeri: these authors analysed NAD + and NADP + from Sigma (purity grade not reported) by HPLC, finding no contamination of NADP + by NAD + within the detection limits of HPLC. The authors, however, point out the presence of  2% NADP + in the NAD + : this was claimed not to intro- duce a bias in their kinetic measurements, and there- fore NAD + was utilized without further purification. In this study, the extremely large effect of  0.3% contamination of the NADP + by the favoured coen- zyme NAD + is directly attributable to the very high level of discrimination between the two coenzymes, so that the 0.3% NAD + produces a rate far higher than that for the 99.7% NADP + . Accordingly, with the mutants, all those in which the discrimination has not been largely abolished give grossly misleading results; only the double-mutant, which approaches dual speci- ficity status, gives a result remotely approaching the truth, because in this situation the 0.3% contamination is at last less dominant. It may be argued that this problem is exceptional, deriving from an extraordinarily high discrimination factor of 80 000. However, because we ourselves assumed until this study that clostridial glutamate dehydrogenase, although NAD + specific, showed a far Table 2. Summary of corrected kinetic parameters for the oxidative deamination. Values of K m and k cat for NADP + were redetermined by utilizing freshly purified NADP + in the enzymatic assays, and are displayed in the table. Discrimination factors are calculated as the ratio k cat ⁄ K m NAD + ⁄ k cat ⁄ K m NADP + . Enzyme NAD + NADP + Discrimination factorK m (mM) k cat (s )1 ) k cat ⁄ K m (s )1 ÆmM )1 ) K m (mM) k cat (s )1 ) k cat ⁄ K m (s )1 ÆmM )1 ) Wild-type 0.11 ± 0.01 22.7 ± 0.5 206 3.2 ± 0.4 8.2 E )3 ±6E )4 0.0026 79 200 F238S 2.21 ± 0.46 19.5 ± 1.9 8.83 1.65 ± 0.11 0.030 ± 0.001 0.018 491 F238S ⁄ P262S 22 ± 3 10.0 ± 0.8 0.45 3.1 ± 0.3 0.283 ± 1.3 E )2 0.091 4.9 M. Capone et al. Coenzyme preference in glutamate dehydrogenase FEBS Journal 278 (2011) 2460–2468 ª 2011 The Authors Journal compilation ª 2011 FEBS 2465 lower level of discrimination, one must wonder how many other examples remain undiscovered of dehydro- genases that are more specific than reported and of protein engineering experiments more successful than the experimenters think. We have carried out a wider programme of mutagenesis at several other positions in the coenzyme binding site and it is clear that all NADP + kinetics will have to be reassessed with freshly purified coenzyme. Experimental procedures Enzymes and substrates The methods for purifying the wild-type glutamate dehy- drogenase and the four mutants from transformed cultures of Escherichia coli have been described in detail elsewhere [22,23,26]. l-Glutamate monosodium salt (99–100%), ammonium chloride (99.5%) and 2-oxoglutarate (97%, 2.3% water) of analytical grade were purchased from Sigma. NAD + lithium salt grade I ( 100%) was obtained from Roche. Different batches of NADP + were: NADP + disodium salt ( 98%), from Roche; NADP + monosodium salt  97%, from Sigma; NADP + monosodium salt > 98%, from Apollo Scientific Ltd. All solutions of the above compounds were freshly prepared in 100 mm phosphate buffer at pH 7.0, and used in enzymatic assays within a few hours. Coenzyme solutions were kept cold and their concentrations deter- mined by measuring A 260 (e NAD ðPÞþ =18· 10 )3 m )1 Æcm )1 ). Examination of coenzyme specificity Kinetic parameters k cat and K m were obtained by measur- ing initial rates of reaction for the mutant and wild-type enzymes in 0.1 m potassium phosphate (pH 6.0, 7.0 and 8.0) with varying concentrations of coenzyme (0.01–2 mm), and l-glutamate fixed at a high concentration (40 mm). Activity was usually measured with a Kontron Uvikon 941 or Cary 50 recording spectrophotometer, thermostatted at 25 °C, by recording changes in A 340 , but in some cases, for greater sensitivity, initial-rate measurements were carried out with a Hitachi F-4500 fluorescence spectrophotometer (Hitachi High-Tech, Tokyo, Japan). A standard curve of the change in fluorescence versus [NAD(P)H] (0.1–1.9 lm) was prepared and enzyme activity was determined by mea- suring the production of NAD(P)H within the linear range. Rapid reaction kinetics An Applied Photophysics SX18.MV-R stopped-flow appa- ratus with a dead-time of 1.3 ms was used for presteady- state kinetic measurements. A 1 mm optical pathlength in a 20 lL cell was used for absorbance measurements at 340 nm; monochromator slit widths were set at 10 nm. The indicated concentrations are final values after mixing, unless stated otherwise. Where possible, l-glutamate, 2-oxogluta- rate and ammonium chloride were used at near-saturating concentrations (40, 20 and 100 mm respectively), as in the steady-state analysis, and at lower concentrations where the reaction in the above conditions was difficult to observe. Coenzyme concentrations were kept at or above K m values derived by steady-state analysis. A minimum of 5 lm enzyme was used for the assays. NADP + purification Coenzymes were analysed with a Waters Controller 600 HPLC system on a reverse-phase column (SUPELCOSIL LC-18-T, particle size 5 lm, 25 · 4.6 cm). The samples were dissolved in 100 mm KH 2 PO 4 and 25–30 ng of each was injected. The elution protocol was as advised by the column manufacturers (Elution Protocol for nucleotides, Supelco Catalogue). Solutions were adjusted to pH 6.0 to prevent damage to the silica solid phase. Data were acquired with a Waters Photodiode Array Detector 996 and chromatograms were monitored at 254 nm. Where indicated, NADP + was purified on a preparative scale (up to 9 mg) using a BioCAD Perseptive System FPLC apparatus with a POROS 20 HQ column (4.60 · 100 mm), a flow rate of 5 mLÆmin )1 and monitoring at 260 and 280 nm. Elution was as follows: 10 mm NaCl isocratic for 10 column volumes; 30 mm NaCl step change and then isocratic for 10 column volumes; gradient increasing to 300 mm NaCl over 50 column volumes. All solutions were double-filtered through 0.2 lm filters before use. Fractions of 2 mL containing NADP + were collected and concentrated by rotary evaporation at 30 °Ctoa volume suitable for gel filtration. NaCl was separated from the concentrated coenzyme solution [30] on a column (2 · 30 cm) of Bio-Gel P2 Fine (45–90 lm wet, Bio-Rad Laboratories, Hercules, CA, USA) at 4 °C. Samples of up to 20 mg NADP + were applied in a volume not exceeding 2.2 mL, and MilliQ-grade water was used for elution, fol- lowed at 254 nm with a BioRad Econo UV Monitor. Des- alting was checked by conductivity measurements on each fraction. The purified NADP + solution was stored at )20 °C and used within 2–3 days of the purification. Impurity peaks from commercial preparations of NADP + were analysed on a mass spectrometer Quattro microÔ (Waters Micromass, Manchester, UK) equipped with electrospray source. Acknowledgements MC was supported by a postgraduate scholarship from the Irish Council for Science, Engineering and Tech- nology. 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