Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis John B. Carrigan and Paul C. Engel School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland An impressive phenomenon in enzymology is the subtle discrimination that nicotinamide nucleotide- dependent enzymes can make between NADP(H) and NAD(H) because the only difference between these two dinucleotide molecules is a phosphate group esteri- fied at the 2¢-OH position of the adenosine ribose [1]. Understanding how enzymes achieve this selectivity is not only of intrinsic scientific interest, but also has practical application. Re-engineering coenzyme speci- ficity is a significant goal, not only simply to minimize coenzyme cost, but also to achieve coenzyme compati- bility in order to couple two enzyme reactions. Predict- ably, the 2¢-phosphate or 2¢-OH interaction site of the enzyme:coenzyme complex has been the focus of initial Keywords coenzyme specificity; glutamate dehydrogenase; NAD(P) + ; nicotinamide nucleotides; site-directed mutagenesis Correspondence P. C. Engel, School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland Fax: +353 1283 7211 Tel: +353 1716 6764 E-mail: paul.engel@ucd.ie (Received 31 May 2007, revised 10 July 2007, accepted 10 August 2007) doi:10.1111/j.1742-4658.2007.06038.x Glutamate dehydrogenase (EC 1.4.1.2–4) from Peptostreptococcus asacch- arolyticus has a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms of k cat ⁄ K m 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 possi- bility 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 dis- crimination against NADPH. By contrast, replacement with a more posi- tively 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 (approxi- mately six-fold) or Asp245 was changed to lysine (32-fold). Coenzyme binding studies confirm that the mutations result in the expected major changes in relative affinities for NADH and NADPH, and pH studies indi- cate that improved affinity for the extra phosphate of NADPH is the pre- dominant reason for the increased catalytic efficiency with this coenzyme. The marked difference between the results of replacing E243 with aspartate and with positive residues implies that the mode of NADPH binding in naturally occurring NADPH-dependent glutamate dehydrogenases differs from that adopted in E243K or E243D and in other dehydrogenases. Abbreviation GDH, L-glutamate dehydrogenase. FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS 5167 redesign efforts, and changes have been made on the basis of 3D comparisons and⁄ or sequence alignments with similar coenzyme-binding structures that selec- tively bind the alternative coenzyme. Frequently, the side chain of a glutamate or aspartate binds the 2¢-OH group and discriminates against NADP + or NADPH. This important residue was highlighted by Wierenga and Hol [2], together with a glycine-rich motif, in a sequence fingerprint that determined coenzyme speci- ficity in the widespread Rossmann fold [3] of dehydro- genases. This key acidic residue has been termed the P7 residue by Baker et al. [4]. Enzymes that exclusively use NADP(H) usually have a smaller, uncharged resi- due at this position and positively charged residues nearby, allowing for better interaction with the 2¢-adenosine phosphate [5]. Protein engineering method- ologies have provided an opportunity to explore the generality of such specificity rules with a number of different dehydrogenases [6–8]. A Rossmann fold is also typical of l-glutamate de- hydrogenases (GDH) (EC 1.4.1.2–4) [9]. GDHs cata- lyse the reversible, nicotinamide-nucleotide-dependent oxidative deamination of l-glutamate to 2-oxoglutarate and ammonia and, in different organisms and meta- bolic circumstances, the reaction may function either to release or to assimilate ammonia [10,11]. Typically, the anabolic reaction is NADPH-dependent, whereas the catabolic reaction uses NAD + , but other members of the family, possibly with amphibolic roles, can use both coenzymes efficiently. Accordingly, the range of GDHs that have been studied cover a complete range of coenzyme specificity, from extreme NAD + specific- ity at one end via varying degrees of dual specificity to extreme NADP + specificity at the other end. An alignment of sequences by Teller et al. [12] dem- onstrated that the GDHs differ from other dehydro- genases in that, surprisingly, the P7 residue is nearly always acidic, regardless of coenzyme specificity. In those enzymes binding NADP(H) only, such as the GDH of Escherichia coli, this residue tends to be aspartate whereas, in NAD(H)- and dual-specific GDHs, the tendency is towards glutamate [12,13]. Figure 1 shows a clear example of the interaction of a P7 glutamate residue with the adenosine ribose of NADH, in this case bound to dual-specificity bovine glutamate dehydrogenase [13]. An exception to this general trend, however, is the NAD(H)-specific GDH from Clostridium symbiosum, which has glycine at the P7 position [3,9,12]. Another NAD(H)-specific GDH in Teller’s align- ment is from Peptostreptococcus asaccharolyticus, also a mesophilic, gram-positive anaerobic bacterium, in which GDH serves the same metabolic role as in C. symbiosum, catalysing the first step in the unusual hydroxyglutarate pathway of glutamate fermentation [14]. Despite this close physiological parallel, the two GDHs show less than 40% sequence identity. The P. asaccharolyticus GDH, which has been purified by a number of groups [15–17], has recently been character- ized in detail in our laboratory following over-expres- sion in E. coli [18]. Like the clostridial GDH, this is an enzyme quite highly specific for NAD(H), with k cat ⁄ K m being approximately 1000-fold greater for NADH than for NADPH, but it conforms to the more general pat- tern of a glutamate residue at the P7 position. This implies that the two enzymes distinguish the cofactors in different ways [3]. In the present study, we have investigated, by means of site-directed mutagenesis and steady-state kinetics, the individual contribution of the P7 glutamate residue, which occupies position 243, as well as the adjacent residues, Trp244 and Asp245, to coenzyme binding and specificity in P. asaccharolyticus GDH. Five mutants were created: in E243K and E243R, the P7 glutamate was replaced by positively charged lysine and arginine, which might stabilize the 2¢-phosphate of NAD(P)H. D245K was created for the same reason. E243D was also constructed because the sequence alignments show aspartate in this position in NADP(H)-specific proteins. The tryptophan residue at position 244 was targeted because removal of this large residue might allow more space for the 2¢-adenosine phosphate, and so it was replaced by serine, which was observed in sequence alignments as the residue most commonly found beside the P7 amino acid of NADP(H) specific GDHs. NADH P1-P6 P7 (Glu) Fig. 1. PYMOL representation of NADH bound to bovine GDH [13]. The P1–P6 region is highlighted in blue and the P7 glutamate, which stabilizes the 2¢-OH of the adenine ribose of the coenzyme, is shown in red stick form. Changing glutamate dehydrogenase coenzyme specificity J. B. Carrigan and P. C. Engel 5168 FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS Results and Discussion Preparation of mutant enzymes All mutants successfully yielded similar quantities of enzyme to the wild-type [18], in the range of 70– 90 mg pure proteinÆL )1 culture. Both the high yield of soluble protein and the fact that all the proteins behaved similarly during ion exchange chromato- graphy suggest that there was no significant over- all perturbation of structure resulting from the mutations. Coenzyme discrimination in the wild-type enzyme As a baseline for these studies, it was necessary to establish the extent of discrimination between the two natural nicotinamide cofactors in the unmutated enzyme. Table 1 shows the separate values of k cat and K m and the derived value for the catalytic efficiency, k cat ⁄ K m . The latter parameter was used to establish discrimination. Thus, the figures of 7.11 s )1 Ælm )1 for NADH and 6.1 · 10 )3 s )1 Ælm )1 for NADPH indicate a 1165-fold discrimination in favour of NADH. This is contributed mainly by a 370-fold difference in K m , with only a 3.1-fold difference in k cat values. Coenzyme specificity of mutants The values for the kinetic parameters at pH 7 (Table 1) reveal improved catalytic efficiency with NADPH as coenzyme for all five mutant proteins, although there is statistical uncertainty with regard to W244S. This improvement is based in all cases on a lowered K m for NADPH. Four of the mutants also showed decreases in k cat values, but these adverse changes were considerably smaller than the favour- able decreases in K m . E243K is the exception in that, as well as having the most dramatic decrease in K m for NADPH, it also demonstrates a 50% increase in k cat . Table 1. Coenzyme specificity of wild-type and mutant Peptostreptococcus asaccharolyticus glutamate dehydrogenases. Initial rates were measured with NADH and NADPH in 100 m M potassium phosphate buffer at pH 7 with 20 mM oxoglutarate and 100 mM ammonium chlo- ride as the fixed concentrations of substrates. Concentrations of NADH and NADPH were varied to obtain values of k cat (column 2) and K m (column 3) for both coenzymes under these conditions. Catalytic efficiency (k cat ⁄ K m ) values (column 4) are used as the basis for comparing each of the mutants with wild-type GDH and also, in each case, for calculating the discrimination factor between the two coenzymes. Thus, column 5 gives the ratio of catalytic efficiency for each mutant enzyme to the corresponding value for the wild-type enzyme with the same coenzyme. This ratio is the ‘factor change’ brought about by the mutation (e.g. for W244S with NADH 1.43 ⁄ 7.11 ¼ 0.201). Column 6 shows another ratio obtained from the catalytic efficiencies in column 4, namely the ratio, for each enzyme, of the catalytic efficiency with NADH to that with NADPH (i.e. the discrimination factor defining the degree of specificity for NADH). This value (i.e. 1170) for the unmutated enzyme is decreased in all the mutants. Column 7 indicates how many folds this discrimination factor is changed in each case. The final column shows, for each mutant, the factor change (e.g. 1170-fold discrimination in the wild-type GDH decreases to 130-fold in E243D, a change by a factor of 8.96). k cat (s )1 ) K m (lM) k cat ⁄ K m s )1 ÆlM )1 Factor change (k cat ⁄ K NADH m ) ⁄ (k cat ⁄ K NADPH m ) Discrimination shift towards NADPH Wild-type NADH 31.3 ± 0.66 4.4 ± 0.45 7.11 1 NADPH 10 ± 1.3 1640 ± 252 6.1 · 10 )3 1 1170 – W244S NADH 10.9 ± 0.23 7.6 ± 0.697 1.43 0.201 NADPH 3.0 ± 0.482 431 ± 88 6.96 · 10 )3 1.14 205 5.68 E243D NADH 16.9 ± 1.33 12.3 ± 3 1.38 0.194 NADPH 5.06 ± 2.13 476 ± 100 1.06 · 10 )2 1.73 130 8.96 E243R NADH 1.71 ± 0.08 38.5 ± 5.5 0.044 6.19 · 10 )3 NADPH 1.67 ± 0.17 38.4 ± 7 0.043 7.05 1.02 1140 E243K NADH 10.6 ± 0.19 36 ± 1.71 0.294 0.041 NADPH 14.8 ± 0.63 53 ± 5 0.28 46 1.05 1110 D245K NADH 16.4 ± 0.16 18 ± 0.99 0.911 0.128 NADPH 6.0 ± 0.44 238 ± 3.4 0.025 4.1 36.4 32 J. B. Carrigan and P. C. Engel Changing glutamate dehydrogenase coenzyme specificity FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS 5169 Table 1 also shows for each mutant enzyme a direct comparison (‘factor change’) of its catalytic efficiency with NADPH with the corresponding figure for the unmutated enzyme. E243K, with a 46.0-fold increase in efficiency compared to wild-type, is by far the best mutant in this respect. Another residue substitution at the same site, E243R, allowed a less dramatic 7.5-fold increase in efficiency, followed by D245K with a 4.1- fold increase and E243D with a 1.73-fold increase. A less definite change was observed with W244S, where an increase in catalytic efficiency of just over 18% with NADPH was measured. For an ideal data set, higher concentrations of NADPH would have been desirable for some of the mutants. However, the high starting absorption of NADPH at concentrations above 0.25 mm made it extremely difficult to measure absorbance changes accurately after enzyme addition. The data obtained nevertheless clearly allow unambiguous ranking of the effects of the various mutations. Accompanying the improved performance with NADPH just described, all mutants showed a marked decrease in efficiency (k cat ⁄ K m ) with NADH as coen- zyme compared to the wild-type (Table 1). E243R shows the most notable shift, being 158-fold worse than the wild-type. The other mutants do not change as much, with W244S and E243D both showing a five-fold decrease and D245K showing a 7.8-fold decrease in efficiency with NADH. The E243K mutant, which shows the most catalytic activity with NADPH, also shows a 24-fold decrease in activity with NADH. The overall discrimination shift from NADH to NADPH for the two positively charged substitutions at P7 is quite similar, with swings of approximately 1100-fold for both E243R and E243K, reflecting the combination of improved efficiency with NADPH and diminished efficiency with NADH. This is much greater than the shift in discrimination values for D245K, E243D and W244S, which range from 32- to 8.96- to 5.68-fold, respectively. The replacement of glu- tamate at the P7 position 243 by the more positively charged lysine and arginine thus gave the most success- ful results in our attempts to alter coenzyme specificity. E243K has a k cat ⁄ K m of 0.28 s )1 Ælm )1 with NADPH, 6.3-fold greater than the value for E243R. Both these mutant enzymes, however, have almost equal activity with the two reduced coenzymes, thus displaying dual specificity. Indeed, the kinetic constants obtained for E243R with the two coenzymes are so strikingly close that it raises at least the possibility of a change in the rate-limiting step to a coenzyme-independent process in the mechanism. The replacement at the same position with Asp pro- duced a much more modest shift in specificity, as seen above, and, even though Asp is widely found at this position in naturally NADP + -dependent GDHs, E243D still shows a preference of over 100-fold for NADH. The replacement of Asp at position 245 with lysine had considerably more effect, decreasing the preference for NADH to a factor of 36. Effect of pH on specific activity Tables 2 and 3 show the specific activity values (lmolÆmg )1 Æmin )1 ) over a range of pH values from 8 to 6 for each enzyme with NADPH and NADH, respectively. Table 3 indicates how all the enzymes show an increase in specific activity with a rise in pH when the coenzyme used is NADH. A similar consis- tent trend was not seen when NADPH was used (Table 2): the wild-type GDH and E243D showed a lowering of specific activity with a pH rise whereas E243K, E243R and W244S showed greater values with increasing pH. It is notable in particular that the two mutants with a positive charge engineered in specifi- cally to engage the negatively charged 2¢-phosphate of NADPH show a substantial increase in activity between pH 6.5 and 7.5, precisely the range over which deprotonation is expected to increase the negative charge on the phosphate. This is most pronounced in the case of the ‘best’ mutant E243K, with almost an Table 2. Specific activity values (lmolÆmg )1 Æmin )1 ) of wild-type and mutant enzymes with 0.1 m M NADPH, 20 mM oxoglutarate and 100 m M ammonium chloride at different pH values. pH 6 pH 6.5 pH 7.0 pH 7.5 pH 8 Wild-type 1.32 1.23 0.73 0.32 0.33 E243K 3.27 9.01 17.6 25.3 20.1 E243R 1.83 2.26 3.05 3.41 4.1 D245K 1.12 2.16 1.53 0.87 1.14 E243D 4.93 5.0 2.98 1.47 1.47 W244S 0.29 0.51 0.33 0.34 0.5 Table 3. Specific activity values (lmolÆmg )1 Æmin )1 ) of wild-type and mutant enzymes with 0.1 m M NADH, 20 mM oxoglutarate and 100 m M ammonium chloride at different pH values. pH 6 pH 6.5 pH 7.0 pH 7.5 pH 8 Wild-type 9.8 20.6 50 155 174 E243K 5.2 9.6 19 56 54 E243R 0.9 2.3 3 12 11 D245K 5.8 12.5 30 68 101.8 E243D 7.2 15 34 90 102 W244S 3.82 6.78 20 56.5 62 Changing glutamate dehydrogenase coenzyme specificity J. B. Carrigan and P. C. Engel 5170 FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS eight-fold increase in rate between pH 6 and pH 7.5. With the other enzymes, including the wild-type GDH, the responses to pH are much smaller with, for exam- ple, less than a two-fold variation over the range 6–8 in D245K and W244S. When interpreting these results, however, it must be noted that the comparison is between rates with fixed substrate concentrations and, in the case of the coenzymes, the fixed concentration will result in widely different degrees of saturation. The rates that are being compared will be dominated therefore to varying degrees by K m . Dissociation constant values with NAD(P)H Dissociation constant (K d ) values (Table 4) were obtained by protein fluorescence. Although binding of a coenzyme by an enzyme may not always be produc- tive, it can at least be unambiguously understood, whereas the physical significance of kinetic parameters is often more complex. (It is important to stress that, although there is evidence for random-order sequen- tial mechanisms for some other GDHs, the detailed kinetic mechanism of this particular GDH has yet to be investigated.) K d values were therefore determined as a direct indication of the effect of the mutations on the enzyme’s binding affinity for reduced coen- zyme. Inner filter effects made it impossible to obtain reliable data with coenzyme concentrations greater than 70 lm and, accordingly, the higher K d values could not be estimated. K d values for the reduced coenzymes were generally much smaller in the pres- ence of 0.5 mm oxoglutarate than without and, indeed, without the other substrate, were generally too high to measure by this method. In most cases where a K d value could be estimated both with and without oxoglutarate, the addition of the second sub- strate tightened coenzyme binding by a factor of at least 20. The exception to this was D245K, which yielded a relatively low K d value (8.6 lm) for NADH even without oxoglutarate. However, in this latter case, the interaction of enzyme and coenzyme caused a very small change in fluorescence. The measurements with NADH in the absence of oxoglutarate show a weakening of binding to varying extents in every one of the mutants. However, with NADPH on its own K d values could not be attained for the wild-type enzyme, nor any of the mutants. Accordingly, the measurements in the presence of oxo- glutarate were the most useful for overall comparison. The wild-type K d for NADH in the presence of oxo- glutarate was 0.72 lm and each of the mutants showed the anticipated weakening of NADH binding, to the extent, in the cases of E243K and E243R, that measur- able interactions with NADH could not be detected over the experimentally accessible range of coenzyme concentration. Conversely, with NADPH, even in the presence of oxoglutarate, a K d value could not be obtained for the wild-type enzyme because the binding is so weak. K d values could not be calculated for W243S either but, in each of the other four mutants, binding of NADPH was tightened sufficiently to give K d values small enough to measure. Of these, E243K gave a value of 37 lm, but E243D, which kinetically was only two-fold improved over wild-type with NADPH, had a much lower K d of 3.5 lm. D245K and E243R, which are more efficient than E243D with NADPH, have much higher K d values of 139 lm and 20.4 lm. This is a striking illustration that tight bind- ing is not necessarily catalytically productive. The strategy behind creating all of these mutants was to try and achieve tighter binding of NADP(H). It can clearly be observed not only that residue 243 (the P7 residue) is a critical residue in the binding of the coenzyme, but also that those residues in its immediate vicinity are of importance. The study of GDH sequence alignments [12] might have suggested that replacement of glutamate with aspartate at this posi- tion would be the best choice. However, the most suc- cessful strategy was to replace Glu243 with a Lys or Arg, stabilizing the adenosine phosphate in the way seen in many other dehydrogenases, and this stabiliza- tion was also achieved, to a lesser extent, by replacing the Asp at 245 with a positively charged amino acid. The fluorescence titrations showed that even with these mutations binding of NADPH was still too weak to allow determination of a K d value in the absence of the other substrate. In the presence of oxoglutarate, however, coenzyme binding was tighter and therefore measurable and, in particular, gave measurable K d values for binding of NADPH to both E243K and E243R, whereas the binding of NADH to these mutants was too weak to measure, in keeping with the Table 4. Dissociation constant values of each enzyme for the reduced coenzyme in the presence and absence of oxoglutarate. Fluorescence titration was carried out on a Perkin-Elmer fluorimeter with excitation set at 290 nm and emission measured at 400 nm. Enzyme K d NADH (lM) (without oxoglutarate) K d NADH (l M) (with oxoglutarate) K d NADPH (l M) (with oxoglutarate) Wild-type 16 ± 4 0.72 ± 0.3 – E243K – – 37 ± 7 E243R – – 20.4 ± 4 D245K 8.57 ± 0.8 4.64 ± 0.57 139 ± 50 E243D 274 ± 20 6.12 ± 1 3.5 ± 0.47 W244S 56.5 ± 10 2.3 ± 0.5 – J. B. Carrigan and P. C. Engel Changing glutamate dehydrogenase coenzyme specificity FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS 5171 fact that these two mutants also gave the highest K m values with NADH. Of the different mutants, E243K is the most success- ful; even though E243R shows a similar shift in dis- crimination in the intended direction, its k cat value with NADPH is decreased relative to the wild-type enzyme, whereas the corresponding figure for E243K (14.8 s )1 ) is not only increased, but is also close to 50% of the k cat of the wild-type enzyme with its natu- ral coenzyme, NADH. Lysine is closer in size to the glutamate it replaces than arginine, and it is possible that the latter is too large to allow optimal orientation of the coenzyme. Although the binding of NADPH is actually tighter to E243R than E243K in the presence of 2-oxoglutarate (Table 4), tighter binding is not necessarily productive binding. To date, high-resolution crystallographic data for P. asaccharolyticus GDH have been elusive [19]. How- ever, even though direct structural studies of coenzyme complexes with these mutants would doubtless help to explain some of these subtle differences more conclu- sively, the main result observed with E243K is not dif- ficult to explain. What still requires further structural insight is, on the one hand, the widespread use of Asp at the P7 position in many naturally occurring NADPH-dependent GDHs and, on the other hand, the failure of the E243D mutation to produce a better result in the present study. It has been suggested [3] that GDHs may normally bind NADPH in the form with only a single charge on the 2¢-phosphate, thus allowing efficient interaction with Asp at P7, although these issues can only be resolved by solution of crystal structures for more binary enzyme coenzyme complexes for GDHs. The present study would seem to support the conclusions of Carrugo and Argos [20], who suggested that discrimination between NAD(H) and NADP(H) is a consequence of the overall proper- ties of the binding pocket and not solely the contribu- tion of a few key residues. What is certain is that the generalizations regarding the P7 position widely assumed to govern NADH ⁄ NADPH specificity cannot be uniformly applied in this enzyme family. Experimental procedures Materials Vectors and bacterial strains for expression and mutagene- sis of P. asaccharolyticus GDH were described previously by Snedecor et al. [21]. In most cases, analytical grade reagents were used. NADH and NADPH, supplied by Roche Diagnostics (Mannheim, Germany) at 98% purity, were used without further purification. l-glutamate (mono- sodium salt), 2-oxoglutarate (monosodium salt) and Q-Sepharose were purchased from Sigma (Poole, UK). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA, USA) and Pfu- turbo DNA polymerase were obtained from Stratagene. Oligonucleotide primers were obtained from Sigma-Genosys (Poole, UK). Expression and purification of the wild-type and mutated P. asaccharolyticus GDH The ptac85 plasmid [12,22], which allows genes to be inserted downstream of the isopropyl thio-b-d-galactoside- inducible tac promoter, was used for the over-expression of wild-type and mutated GDH genes in E. coli TG1. PCR overlap extension and whole plasmid synthesis were used to generate point mutations. Transformation of the E. coli TG1 host, growth, induction, harvesting, breakage and enzyme purification were as described by Carrigan et al. [18]. This involved utilizing the thermostability of the enzyme by heating the preparation to 70 °C before binding to an ion exchange column. SDS ⁄ PAGE gels were used to check that the over-expressed protein was soluble. Determination of kinetic parameters Apparent values of k cat and K m for the coenzyme, either NADH or NADPH, at fixed concentrations of the other two substrates (20 mm 2-oxoglutarate and 100 mm ammo- nium chloride), were determined for the reductive amina- tion reaction in 0.1 m potassium phosphate buffer at pH 7. Concentrations of NAD(P)H were varied between 0.001 and 0.4 mm. Reaction rates at 25 °C after addition of the enzyme were monitored on a Cary 50 recording spectro- photometer (Varian Inc., Palo Alto, CA, USA) by measur- ing the decrease in NAD(P)H concentration via the change in A 340 nm , using an extinction coefficient of 6220 m )1 Æcm )1 for both NADH and NADPH [23]. For the lowest coen- zyme concentrations, the more sensitive Hitachi 1500 fluo- rimeter (Hitachi Corp., Tokyo, Japan) was used, with excitation wavelength set at 340 nm and emission at 460 nm. K m and V max values were obtained using enzpack 3.0 (Biosoft, Great Shelford, UK), which also generated a Wilkinson error value [24]. All reaction rates were measured at least in duplicate, usually with an agree- ment within 2–3%, and enzyme concentrations were adjusted to ensure that these measurements could be confi- dently made over the very wide activity range explored. Thus, where activity was high (e.g. wild-type GDH with NADH), the concentration of enzyme was kept low enough to obtain good initial linearity; where activity was low (e.g. wild-type GDH with NADPH), the plentiful supply of pure enzyme meant that large amounts could be added to pro- duce a high enough rate for accurate measurement. Changing glutamate dehydrogenase coenzyme specificity J. B. Carrigan and P. C. Engel 5172 FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS Measurement of K d Fluorescence titration was carried out on a Perkin-Elmer fluorimeter (Perkin-Elmer Life Sciences, Boston, MA, USA) with excitation set at 290 nm and emission measured at 400 nm. The protein emission peak is actually at 340 nm, but light absorption by the reduced coenzyme at this wavelength could cause experimental error. Protein (20 lg) was added to varying concentrations of NAD(P)H in 0.1 m potassium phosphate buffer at pH 7 and at a con- stant temperature of 25 °C. An attempt was made to obtain values in the presence of 0.5 mm oxoglutarate as well as without. The changes in fluorescence of the protein were plotted versus coenzyme concentration. The data were fitted to a saturation plot using enzpack which provided an esti- mate of the K d . Acknowledgements This study was supported in part by a Basic Science research grant SC2002 ⁄ 0502 from Enterprise Ireland and this assistance is gratefully acknowledged. We also wish to thank Roche Diagnostics and the Irish Ameri- can Partnership for studentship support in the initial stages of the project and the EU for making it possible through the Marie Curie scheme for J. B. Carrigan to spend a valuable period in the laboratory of Professor Janet Thornton at Cambridge. References 1 Warburg O, Christian W & Griese A (1935) Wasserst- offu ¨ bertragendes Co-Ferment, seine Zusammensetzung und Wirkungsweise. Biochem Z 282, 157–198. 2 Wierenga RK & Hol WG (1983) Predicted nucleotide- binding properties of p21 protein and its cancer-associ- ated variant. Nature 302, 842–844. 3 Baker PJ, Britton KL, Rice DW, Rob A & Stillman TJ (1992) Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold. Implications for nucleotide specificity. J Mol Biol 228, 662–671. 4 Rossmann MG, Moras D & Olsen KW (1974) Chemical and biological evolution of nucleotide-binding protein. Nature 250, 194–199. 5 Wierenga RK, Terpstra P & Hol WG (1986) Prediction of the occurrence of the ADP-binding bab-fold in pro- teins, using an amino acid sequence fingerprint. J Mol Biol 187, 101–107. 6 Feeney R, Clarke AR & Holbrook JJ (1990) A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme. Biochem Biophys Res Commun 166, 667–672. 7 Chen Z, Tsigelny I, Lee WR, Baker ME & Chang SH (1994) Adding a positive charge at residue 46 of Dro- sophila alcohol dehydrogenase increases cofactor speci- ficity for NADP + . FEBS Lett 356, 81–85. 8 Scrutton NS, Berry A & Perham RN (1990) Redesign of the coenzyme specificity of a dehydrogenase by pro- tein engineering. Nature 343, 38–43. 9 Baker PJ, Britton KL, Engel PC, Farrants GW, Lilley KS, Rice DW & Stillman TJ (1992) Subunit assembly and active site location in the structure of glutamate dehydrogenase. Proteins Struct Funct Gen 12, 75–86. 10 Smith EL, Austen BM, Blumenthal KM & Nyc JF (1975) Glutamate dehydrogenases. In The Enzymes, Vol. 11 (Boyer PD, ed), pp. 293–367. Academic Press, New York, NY. 11 Hudson RC & Daniel RM (1993) L-glutamate dehydro- genases: distribution, properties and mechanism. Comp Biochem Physiol B 106, 767–792. 12 Teller JK, Smith RJ, McPherson MJ, Engel PC & Guest JR (1992) The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli. Eur J Biochem 206, 151–159. 13 Smith TJ, Peterson PE, Schmidt T, Fang J & Stanley CA (2001) Structures of bovine glutamate dehydroge- nase complexes elucidate the mechanism of purine regu- lation. J Mol Biol 320, 707–720. 14 Buckel W & Barker HA (1974) Two pathways of gluta- mate fermentation by anaerobic bacteria. J Bacteriol 117, 1248–1260. 15 Kew OM & Woolfolk CA (1970) Preparation of gluta- mate dehydrogenase from Micrococcus aerogenes. Biochem Biophys Res Commun 39, 1126–1133. 16 Johnson WM & Westlake DW (1972) Purification and characterization of glutamic acid dehydrogenase and ketoglutaric acid reductase from Peptococcus aerogenes. Can J Microbiol 18, 881–892. 17 Hornby DP & Engel PC (1984) Characterization of Peptostreptococcus asaccharolyticus glutamate dehydro- genase purified by dye-ligand chromatography. J Gen Microbiol 130, 2385–2394. 18 Carrigan JB, Coughlan S & Engel PC (2005) Proper- ties of the thermostable glutamate dehydrogenase of the mesophilic anaerobe Peptostreptoccus asaccharolyti- cus purified by a novel method after over-expression in an Escherichia coli host. FEMS Microbiol Lett 244, 53–59. 19 Waugh ML (1994) X-ray crystallographic studies on the catalytic mechanism of glutamate dehydrogenase. PhD Thesis, University of Sheffield, UK. 20 Carrugo O & Argos P (1997) NADP-dependent enzymes. I: Conserved stereochemistry of cofactor bind- ing. Proteins 28, 10–28. J. B. Carrigan and P. C. Engel Changing glutamate dehydrogenase coenzyme specificity FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS 5173 21 Snedecor B, Chu H & Chen E (1991) Selection, expres- sion, and nucleotide sequencing of the glutamate dehy- drogenase gene of Peptostreptococcus asaccharolyticus. J Bacteriol 173, 6162–6167. 22 Marsh P (1986) Ptac-85, an E. coli vector for expression of non-fusion proteins. Nucleic Acids Res 14, 3603. 23 Kaplan NO, Ciotti MM & Stolzenbach FE (1957) Stud- ies on the interaction of diphosphopyridine nucleotide analogs with dehydrogenases. Arch Biochem Biophys 69, 441–457. 24 Wilkinson GN (1961) Statistical estimations in enzyme kinetics. Biochem J 80, 324–332. Changing glutamate dehydrogenase coenzyme specificity J. B. Carrigan and P. C. Engel 5174 FEBS Journal 274 (2007) 5167–5174 ª 2007 The Authors Journal compilation ª 2007 FEBS . examined the possi- bility of altering coenzyme specificity of the Peptostreptococcus enzyme, and, more specifically, the role of residue 243 and neighbouring residues in coenzyme binding, by introducing. Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis John B. Carrigan and Paul C. Engel School of Biomolecular. in the absence of the other substrate. In the presence of oxoglutarate, however, coenzyme binding was tighter and therefore measurable and, in particular, gave measurable K d values for binding