Glutamate dehydrogenases: the why and how of coenzyme specificity.

NAD(+) and NADP(+), chemically similar and with almost identical standard oxidation-reduction potentials, nevertheless have distinct roles, NAD(+) serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD(+)-dependent for glutamate oxidation, NADP(+)-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD(+) reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD(+) but maintains equilibrium using the same pool as accessed by ß-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP(+) reduction by NADH, maintaining the coenzyme pools at different oxidation-reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD(+)-dependent, NADP(+)-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD(+) or for NADP(+) has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2'- and 3'-hydroxyls, dictating NAD(+) specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD(+) only, NADP(+) only, or in higher animals both.

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity.

Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotide-binding domain have been linked to NAD(+) vs. NADP(+) specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD(+)-specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD(+) in Domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD(+) binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD(+) vs. NADP(+) recognition.

Molecular recruitment as a basis for negative dominant inheritance? propagation of misfolding in oligomers of IMPDH1, the mutated enzyme in the RP10 form of retinitis pigmentosa.

Retinitis pigmentosa, causing progressive blindness, is genetically heterogeneous. RP10, due to a defect in inosine monophosphate dehydrogenase 1 (IMPDH1), shows autosomal dominant inheritance. Recombinantly expressed clinical mutants show unaltered kinetic behaviour. It is unclear why reportedly impaired DNA binding is important and how it would explain negative dominance. An alternative view relates to the mutant proteins' tendency to aggregate. Regarding negative dominance, a key question is whether the defective protein can subvert the function of its normal counterpart in the same cell. Potentially, the homotetrameric structure of IMPDH1 might offer a vehicle for such an effect. We have established a reliable protocol for reproducible refolding of recombinantly expressed IMPDH1 in vitro. Clinical mutants R224P and D226N both show impaired folding. For equimolar mixtures of normal and mutant enzymes, independent refolding would predict activity regain midway between pure mutant and pure normal. Under various conditions regain is close to the mutant figure, suggesting that, in hybrid tetramers, mutant subunits impose their faulty conformation on normal partners. The observed molecular recruitment is a negative counterpart of the intra-allelic complementation, also mediated via oligomeric structure and postulated many years ago by Fincham. These findings appear potentially to account for the negative dominant inheritance. This interpretation must be provisional at present, as the predominant transcript in retina is an alternatively spliced version not fully identical to that used in our study. The results nevertheless have a general significance in pointing to a mechanism for negative dominance that could be widespread.

Development of a satisfactory and general continuous assay for aminotransferases by coupling with (R)-2-hydroxyglutarate dehydrogenase.

A continuous general spectrophotometric assay for measuring the activity of aminotransferases has been developed. It is based on the transamination of a keto compound (amino acceptor) and l-glutamate (amino donor), yielding the corresponding amino compound and 2-oxoglutarate. The rate of formation of 2-oxoglutarate is measured in a coupled reaction with overproduced recombinant nicotinamide adenine dinucleotide (NAD(+))-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans, with the rate of absorbance decrease at 340nm indirectly reflecting the aminotransferase activity. This new method allows continuous monitoring of the course of transamination. Because glutamate and 2-oxoglutarate are obligatory participants in most biological transamination reactions, a coupled assay based on measuring the formation of 2-oxoglutarate has very wide applicability. The article demonstrates its utility with branched-chain amino acid aminotransferase and l-valine:pyruvate aminotransferase.

Overexpression in a non-native halophilic host and biotechnological potential of NAD+-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014.

Enzymes produced by halophilic archaea are generally heat resistant and organic solvent tolerant, and accordingly important for biocatalytic applications in 'green chemistry', frequently requiring a low-water environment. NAD(+)-dependent glutamate dehydrogenase from an extremely halophilic archaeon Halobacterium salinarum strain NRC-36014 was selected to explore the biotechnological potential of this enzyme and genetically engineered derivatives. Over-expression in a halophilic host Haloferax volcanii provided a soluble, active recombinant enzyme, not achievable in mesophilic Escherichia coli, and an efficient purification procedure was developed. pH and salt dependence, thermostability, organic solvent stability and kinetic parameters were explored. The enzyme is active up to 90 °C and fully stable up to 70 °C. It shows good tolerance of various miscible organic solvents. High concentrations of salt may be substituted with 30 % DMSO or betaine with good stability and activity. The robustness of this enzyme under a wide range of conditions offers a promising scaffold for protein engineering.

Making biochemistry count: life among the amino acid dehydrogenases.

The guiding principle of the IAS Medal Lecture and of the research it covered was that searching mathematical analysis, depending on good measurements, must underpin sound biochemical conclusions. This was illustrated through various experiences with the amino acid dehydrogenases. Topics covered in the present article include: (i) the place of kinetic measurement in assessing the metabolic role of GDH (glutamate dehydrogenase); (ii) the discovery of complex regulatory behaviour in mammalian GDH, involving negative co-operativity in coenzyme binding; (iii) an X-ray structure solution for a bacterial GDH providing insight into catalysis; (iv) almost total positive co-operativity in glutamate binding to clostridial GDH; (v) unexpected outcomes with mutations at the catalytic aspartate site in GDH; (vi) reactive cysteine as a counting tool in the construction of hybrid oligomers to probe the basis of allosteric interaction; (vii) tryptophan-to-phenylalanine mutations in analysis of allosteric conformational change; (viii) site-directed mutagenesis to alter substrate specificity in GDH and PheDH (phenylalanine dehydrogenase); and (ix) varying strengths of binding of the 'wrong' enantiomer in engineered mutant enzymes and implications for resolution of racemates.

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined.

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-ß-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.

Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development.

BACKGROUND: Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target. METHODS: The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated. RESULTS: No growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10Δgdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of α-ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites. CONCLUSIONS: First, the data support the conclusion that D10Δgdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic parasite development.

Clinical mutants of human glucose 6-phosphate dehydrogenase: impairment of NADP(+) binding affects both folding and stability.

Human glucose 6-phosphate dehydrogenase (G6PD) has both the "catalytic" NADP(+) site and a "structural" NADP(+) site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PD(Wisconsin) (R393G) and G6PD(Nashville) (R393H), and G6PD(Fukaya) (G488S) and G6PD(Campinas) (G488V), in which the mutations are in the vicinity of the "structural" NADP(+) site, showed elevated K(d) values of the "structural" NADP(+), ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP(+) and DTT at 25 degrees C. The refolding yields of the mutants exhibited strong NADP(+)-dependence and ranged from 1.5% to 59.4% with 1000 microM NADP(+), in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of "structural" NADP(+) can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state.

Crystallization and preliminary structural analyses of glutamate dehydrogenase from Peptoniphilus asaccharolyticus.

Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptoniphilus asaccharolyticus has been expressed as a selenomethionine-derivatized recombinant protein and diffraction-quality crystals have been grown that are suitable for structure determination. Preliminary structural analyses indicate that the protein assembles as a homohexameric enzyme complex in solution, similar to other bacterial and mammalian enzymes to which its sequence identity varies between 25 and 40%. The structure will provide insight into its preference for the cofactor NADH (over NADPH) by comparisons with the known structures of mammalian and bacterial enzymes.

An NSAID-like compound, FT-9, preferentially inhibits gamma-secretase cleavage of the amyloid precursor protein compared to its effect on amyloid precursor-like protein 1.

Inhibition of gamma-secretase cleavage of the amyloid precursor protein (APP) is a prime target for the development of therapeutics for treating Alzheimer's disease; however, complete inhibition of this activity would also impair the processing of many other proteins, including the APP homologues, amyloid precursor-like protein (APLP) 1 and 2. To prevent unwanted side effects, therapeutically useful gamma-secretase inhibitors should specifically target APP processing while sparing cleavage of other gamma-substrates. Thus, since APLP1 and APLP2 are more similar to APP than any of the other known gamma-secretase substrates and have important physiological roles in their own right, we reasoned that comparison of the effect of gamma-secretase inhibitors on APLP processing should provide a sensitive indicator of the selectivity of putative inhibitors. To address this issue, we have optimized microsome and cell culture assays to monitor the gamma-secretase proteolysis of APP and APLPs. Production of the gamma-secretase-generated intracellular domain (ICD) occurs more rapidly from APLP1 than from either APLP2 or APP, suggesting that APLP1 is a better gamma-substrate and that substrate recognition is not restricted to the highly conserved amino acid sequences surrounding the epsilon-site. As expected, the well-characterized gamma-secretase modulator, fenofibrate, did not inhibit ICD release, whereas a related compound, FT-9, inhibited gamma-secretase both in microsomes and in whole cells. Importantly, FT-9 displayed a preferential effect, inhibiting cleavage of APP much more effectively than cleavage of APLP1. These findings suggest that selective inhibitors can be developed and that screening of compounds against APP and APLPs should assist in this process.

Modular coenzyme specificity: a domain-swopped chimera of glutamate dehydrogenase.

Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD(+)-specific) and Escherichia coli (EcGDH) (NADP(+)-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain I) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V(max)) in both directions than either of the parent enzymes from which it is constructed.

An optimised system for refolding of human glucose 6-phosphate dehydrogenase.

BACKGROUND: Human glucose 6-phosphate dehydrogenase (G6PD), active in both dimer and tetramer forms, is the key entry enzyme in the pentose phosphate pathway (PPP), providing NADPH for biosynthesis and various other purposes, including protection against oxidative stress in erythrocytes. Accordingly haemolytic disease is a major consequence of G6PD deficiency mutations in man, and many severe disease phenotypes are attributed to G6PD folding problems. Therefore, a robust refolding method with high recovery yield and reproducibility is of particular importance to study those clinical mutant enzymes as well as to shed light generally on the refolding process of large multi-domain proteins. RESULTS: The effects of different chemical and physical variables on the refolding of human recombinant G6PD have been extensively investigated. L-Arg, NADP+ and DTT are all major positive influences on refolding, and temperature, protein concentration, salt types and other additives also have significant impacts. With the method described here, ~70% enzyme activity could be regained, with good reproducibility, after denaturation with Gdn-HCl, by rapid dilution of the protein, and the refolded enzyme displays kinetic and CD properties indistinguishable from those of the native protein. Refolding under these conditions is relatively slow, taking about 7 days to complete at room temperature even in the presence of cyclophilin A, a peptidylprolyl isomerase reported to increase refolding rates. The refolded protein intermediates shift from dominant monomer to dimer during this process, the gradual emergence of dimer correlating well with the regain of enzyme activity. CONCLUSION: L-Arg is the key player in the refolding of human G6PD, preventing the aggregation of folding intermediate, and NADP+ is essential for the folding intermediate to adopt native structure. The refolding protocol can be applied to produce high recovery yield of folded protein with unaltered properties, paving the way for future studies on clinical G6PD mutants with folding defects and providing a useful model system to study the folding process of oligomeric proteins.

Homotropic allosteric control in clostridial glutamate dehydrogenase: different mechanisms for glutamate and NAD+?

Clostridial glutamate dehydrogenase mutants with the 5 Trp residues in turn replaced by Phe showed the importance of Trp 64 and 449 in cooperativity with glutamate at pH 9. These mutants are examined here for their behaviour with NAD+ at pH 7.0 and 9.0. The wild-type enzyme displays negative NAD+ cooperativity at both pH values. At pH 7.0 W243F gives Michaelis-Menten kinetics, and the same behaviour is shown by W243F and also W310F at pH 9.0, but not by W64F or W449F. W243 and W310 are apparently much more important than W64 and W449 for the coenzyme negative cooperativity, implying that different conformational transitions are involved in cooperativity with the coenzyme and with glutamate.

Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli.

The gene for Escherichia coli glutamate dehydrogenase (EcGDH) has been overexpressed, and a simplified purification procedure afforded greatly increased yields of c. 40 mg pure EcGDH L(-1) culture. EcGDH was unstable at a low concentration in plastic tubes, but stabilization measures allowed a robust kinetic characterization. Contrary to past reports, EcGDH deviates from Michaelis-Menten kinetics, exhibiting apparent mild negative co-operativity with both l-glutamate and NADP+, with Hill coefficients of 0.90 and 0.92, respectively. NADPH yielded simple Michaelis-Menten kinetics but both 2-oxoglutarate and NH4+ showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.

Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the D-enantiomer from racemic mixtures.

With a view to their use in the kinetic resolution of racemic non-natural amino acids, five variants of the enzyme L-phenylalanine dehydrogenase, the wild-type enzyme from Bacillus sphaericus and four active-site mutants, have been tested with a range of amino acids. In each case, the rates of reaction with 0.2 mM L-amino acid and with the racemic mixture at 0.4 mM were compared, so that the starting concentration of the active substrate was kept constant. Although the D-amino acids are not substrates, they were inhibitory in all cases. The extent of inhibition, however, varied greatly from compound to compound and among the mutants. With the N145L mutant and DL 4-O-Me-Phe, the equimolar D-enantiomer gave 83.2% inhibition, and with the wild-type enzyme there was 86.7% inhibition with racemic norleucine. By contrast, with these same substrates the N145V mutant showed less than 9% and 24% inhibition respectively. The N145A mutant was selected for use with DL-4-Cl-Phe. The pH was decreased from the enzyme's optimum of 10.4 to 9.5 to minimise breakdown of the coenzyme NAD(+), and the coenzyme was recycled by molecular oxygen with the assistance of a commercial diaphorase. Reaction on a 200 micromole scale in 20 ml ethanolamine HCl buffer, pH 9.5, with 25 microg N145A enzyme and 100 microg diaphorase, was monitored by chiral HPLC. The L-isomer was removed to an extent of >99% after 40 h, with the D-isomer peak undiminished. The pure D-isomer was isolated from the reaction mixture in 85% overall yield after ion-exchange chromatography.

Functional properties of two mutants of human glucose 6-phosphate dehydrogenase, R393G and R393H, corresponding to the clinical variants G6PD Wisconsin and Nashville.

Two severe Class I human glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49) mutations, G6PD(Wisconsin) (nt1177 C-->G, R393G) and G6PD(Nashville) (nt1178 G-->A, R393H), affect the same codon, altering a residue in the dimer interface close to the "structural" NADP+ site. These mutations are predicted to influence interaction with the bound "structural" NADP+, long supposed to be crucial for enzyme stability. Recombinant proteins corresponding to these mutants have been constructed, expressed and purified to homogeneity. Steady-state kinetic parameters of the mutant enzymes were comparable to those of normal human G6PD, indicating that the mutations do not alter catalytic efficiency drastically. However, investigations of thermostability, urea denaturation, protease digestion, and hydrophobic exposure demonstrated that G6PD R393H is less stable than normal G6PD or R393G, and stability was more NADP+-dependent. Apoenzymes were prepared by removal of "structural" NADP+. Again the G6PD(Nashville) protein was markedly less stable, and its dissociation constant for "structural" NADP+ is approximately 500 nM, about 10 times higher than values for R393G (53 nM) and normal G6PD (37 nM). These results, together with structural information, suggest that the instability of the R393H protein, enhanced by the weakened binding of "structural" NADP+, is the likely cause of the severe clinical manifestation observed for G6PD(Nashville). They do not, however, explain the basis of disease in the case of G6PD(Wisconsin).

Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis.

Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptostreptococcus asaccharolyticus 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-specific 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 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 indicate that improved affinity for the extra phosphate of NADPH is the predominant 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.

The contribution of tryptophan residues to conformational changes in clostridial glutamate dehydrogenase--W64 and W449 as mediators of the cooperative response to glutamate.

The hexameric glutamate dehydrogenase of Clostridium symbiosum has previously been shown to undergo a pH-dependent inactivating conformational change that perturbs the environment of one or more Trp residues and is reversed by glutamate in a highly cooperative fashion with a Hill coefficient of almost 6. Five single mutants have now been made in which each of the Trp residues in turn has been replaced by Phe. All five were successfully over-produced as soluble proteins and purified. Far-UV CD showed that none of the mutations significantly affected secondary structure. All five proteins were active, ranging from 13 U.mg(-1) (W64F) to 20.8 U.mg(-1) (W393F), compared to 20 U.mg(-1) for wild-type, and the kinetic parameters at pH 7 were little changed, except for a five- to six-fold increase in Km for glutamate in W243F. Thermostability was also relatively little changed, although W310F and W393F were somewhat more stable and W64F less stable than the unmutated enzyme. All still showed the characteristic reversible, time-dependent high-pH inactivation. Near-UV CD spectra, reflecting the environment of aromatic residues, were recorded at both pH 7 and 8.8, and four of the mutants showed essentially the same perturbation in the 280 nm region as the wild-type enzyme. W64F, however, showed essentially no change. W64 is thus clearly a passive reporter of the pH-dependent conformational change, and not actually required for the transition to occur. The CD comparisons also suggest that the aromatic CD spectrum is contributed almost entirely by W64 and W449. Consistent with the pH-dependent change, all five mutant proteins also showed a positively cooperative response to glutamate at pH 9, reversing the inactivation. However, the Hill coefficient decreased from > 5 for wild-type to approximately 3 for the active site cleft mutation W243F and to approximately 2 for the interfacial mutants W64F and W449F in which the trimer-trimer interaction may be directly interrupted. W64 of each subunit is in contact with W449 in its dimer partner at the trimer-trimer interface. It seems that, although neither of these two residues is required for the pH-dependent change, together, they are essential in mediating the total cooperativity of the hexameric enzyme's response to glutamate and are presumably directly involved in transmitting conformational information between the two trimers.