Binding studies of a spin-labelled oxidized coenzyme to bovine-liver glutamate dehydrogenase.

NAD+ with a nitroxide piperidine ring linked to the NH2 group of the adenine possesses full coenzymatic activity with glutamate dehydrogenase. Electron spin resonance spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin-label. Binding studies in phosphate buffer demonstrate: (a) weak binary binding to the enzyme with a dissociation constant in the order of 2mM;(b) an indication for negative cooperativity or different sites for binding to enzyme-2-oxoglutarate, with dissociation constants in the order of 20--250muM; (c) similar but much weaker binding to enzyme-2-oxoglutarate-ADP; (d) a strong positive cooperative binding to enzyme-2-oxoglutarate-GTP, dependent on the enzyme concentration. Binding of phosphate to the enzyme with a Kd of about 20 mM or binding of pyrophosphate or tripolyphosphate with a Dd of about 2.5 mM enhances the binding of spin-labelled NAD+ in the presence of 2-oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.     

Conformational changes in bovine-liver glutamate dehydrogenase: a spin-label study.

A spin-labelled analogue of p-chloromercuribenzoate reacts specifically with glutamate dehydrogenase. The most marked change in the properties of the spin-labelled enzyme is a fivefold decrease in the rate of reduction of the coenzyme by L-glutamate and no change in the rate of oxidation by 2-oxoglutarate. The electron spin resonance spectrum is a sensitive probe for the conformational state of the enzyme. Spin-labelled glutamate dehydrogenase in the presence of saturating concentrations of NADPH and 2-oxoglutarate or L-glutamate shows a complete conformational change while in the presence of NADP+ and 2-oxoglutarate only half of the protomers have changed conformation. The conformational change upon addition of NADPH to the spin-labelled glutamate dehydrogenase in the presence of 2-oxoglutarate happens in a concerted way between 20 and 80% saturation with NADPH. One of the conformations is favoured by the activator ADP while the other is favoured by the inhibitor GTP.     

The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.     

Relaxation kinetic studies of coenzyme binding to glutamate dehydrogenase from beef liver.

The enzyme, D-erythrodihydroneopterin triphosphate synthetase from rat brain was observed to have a significantly lower specific activity than that from liver due to their degree of dephosphorylation during preparation. The brain enzyme could be phosphorylated $\text{in vitro}$ in presence of [³²P]-ATP and protein kinase, resulting in an increased specific activity. Isolation of brain enzyme in presence of 0.8 M NaF allowed recovery of the enzyme phosphorylated at residue 67 (serine) as determined by a new assay for phosphate. This enzyme is present in synaptosomes and its state of phosphorylation may regulate the rate at which dihydrobiopterin, the precursor of the hydroxylase cofactor (tetrahydrobiopterin, BH₄), is synthesized by synaptosomes.     

Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase

Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD(+) binding site within human GDH encoded by the synthetic human GDH gene and expressed in Escherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+) binding site. Amino acid sequencing and compositional analysis identified Glu(279) as the site of photoinsertion into human GDH, suggesting that Glu(279) is located at or near the NAD(+) binding site. The importance of the Glu(279) residue in the binding of NAD(+) was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu(279) has no effects on the expression or stability of the different mutants. The K(m) values for NAD(+) were 10-14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V(max) values were similar for wild-type and mutant GDHs. The efficiency (k(cat)/K(m)) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K(m) values for NAD(+). In contrast to the K(m) values for NAD(+), wild-type and mutant GDHs show similar K(m) values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu(279) is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu(279) plays an important role for efficient binding of NAD(+) to human GDH.     

Structural organization of a hexamer of glutamate dehydrogenase. 3. Effect of the coenzyme and substrate on the urea-induced dissociation and inactivation of the hexamer

Effects of coenzyme (NADH) and substrate (2-oxoglutarate) on the urea-induced dissociation and inactivation of immobilized phosphopyridoxyl derivative of bovine liver glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) have been studied. Urea at concentration 3.0 to 4.0 M in the presence of NADH induced dissociation of the enzyme's hexamer to catalytically inactive immobilized dimer. In the presence of both NADH and 2-oxoglutarate at the urea concentration 1.0 to 2.0 M the hexamer dissociated to the conformationally stable immobilized trimer possessing 60% catalytic activity of the hexamer. Studies of regulatory properties of the immobilized trimer showed that the allosteric inhibition of glutamate dehydrogenase by GTP was realized on the level of trimers, where the subunits interact through identical heterological contacts.     

Human erythrocyte glucose 6-phosphate dehydrogenase. Content of bound coenzyme

A series of peptide analogs of luteinizing hormone releasing hormone (LH-RH), altered at position 6 and 10, was synthesized and evaluated $\text{in vivo}$ for the ability to induce ovulation in the diestrous rat and $\text{in vitro}$ for ability to release pituitary luteinizing hormone and follicle stimulating hormone. All the analogs with D-amino acid substitutions at position 6, even those with large bulky side chain, exhibited an amazingly high potency compared with the parent hormone, LH-RH. On the basis of the biological activities, structure-activity relationships in the central part of this molecule were discussed in detail.     

Inhibition of glutamate dehydrogenase and malate dehydrogenases by palmitoyl coenzyme A.

In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA.     

Spin-labeling studies of beef-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase was spin labeled with a nitroxide derivative of parachloromercuribenzoate. The ESR spectrum was of the immobilized type and the labeling yield 0.6 mole of spin label bound per mole of protomer under standard conditions. The specific activity of the labeled enzyme was not modified but the activation by ADP abolished. Inhibition by GTP was not altered but the ESR spectrum showed that the bound spin label was further immobilized in the presence of GTP and NADPH. In the presence of the coenzyme NADPH, the labeling yield decreased to half its initial value. Such a protection effect was observed neither with NADH nor with ADP.     

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH [( S]NADPH). For native enzyme, ultrafiltration studies revealed that [S]NADPH reversibly occupies about two sites/enzyme subunit in the absence of other ligands; by the addition of ADP, [S]NADPH binding can be limited to one molecule/subunit. The enzyme is irreversibly inactivated by reaction with 4-(iodoacetamido)salicylic acid (ISA) at lysine126 within the 2-oxoglutarate binding site [Holbrook, J.J., Roberts, P.A. & Wallis, R.B. (1973) Biochem. J. 133, 165-171]. ISA-modified enzyme binds 1 molecule [S]NADPH/subunit in the absence of ADP, suggesting that reaction at the substrate site blocks binding at the catalytic, but not at the regulatory site. The fluorescence spectrum of ISA-modified enzyme overlaps the absorption spectrum of [S]NADPH allowing a distance measurement between these sites by resonance energy transfer. [S]NADPH quenches the emission of ISA-modified enzyme, yielding 3.2 nm as the average distance between sites. ADP competes for the [S]NADPH site but does not affect the fluorescence of ISA-modified enzyme, indicating that [S]NADPH quenching is attributable to energy transfer rather than to a conformational change. The 3.2 nm thus represents the distance between the 2-oxoglutarate and reduced coenzyme regulatory sites of glutamate dehydrogenase.     

Conformation of coenzyme fragments when bound to lactate dehydrogenase

The conformations of adenosine, 5′-AMP and 5′-ADP when bound to dogfish M₄ lactate dehydrogenase at pH 7.8 or greater have been determined at 2.8 Å resolution to investigate the events on coenzyme binding. The coenzyme fragments AMP and ADP induce a conformational change in lactate dehydrogenase at pH values less than 6.0 in the same way as do NAD⁺, NADH or ADPR at any pH value. The structure of NAD⁺ when bound to lactate dehydrogenase had previously been determined at 5.0 Å resolution. The structures of the bound adenosine, AMP, ADP and NAD⁺ are compared with the preliminary structure of NAD in a 3.0 Å resolution map of the ternary complex LDH-NAD—pyruvate. Small but significant changes in the binding of the phosphates could be important in the folding of the protein loop over the substrate binding pocket.     

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.     

Alpha-isoenzyme of alcohol dehydrogenase from monkey liver. Cloning, expression, mechanism, coenzyme, and substrate specificity.

The cDNA for the alpha-isoenzyme from rhesus monkey (Macaca mulatta) liver was cloned and expressed in yeast. The alpha-isoenzymes of human and monkey liver alcohol dehydrogenase differ from the other human and horse liver enzymes in having Met57, Ala93, and Val116 instead of Leu57, Phe93, and Leu116 in the substrate binding pocket and Gly47 instead of Arg47 near the pyrophosphate moiety of the coenzyme. The effects of these differences on the kinetic mechanism, substrate specificity, and coenzyme binding were studied with the purified, recombinant monkey alpha-isoenzyme (MmADH alpha) and mutated enzymes with Gly47 substituted with His or Arg. The mechanism appears to be random for the binding of NAD+ and ethanol and ordered for NADH and acetaldehyde, with formation of a dead-end enzyme-NADH-ethanol complex. MmADH alpha reacts 130-fold slower (V/K) with ethanol and 3-25-fold slower with 2-methyl alcohols but 20-fold faster with cyclohexanol, as compared with horse (Equus caballus) liver EE isoenzyme (EqADH). MmADH alpha is stereoselective for the R isomer of 2-butanol, whereas EqADH favors the S isomer. Both enzymes have comparable reactivity with larger primary alcohols. MmADH alpha is more reactive with secondary alcohols and has highest activity with cyclohexanol. However, it does not react with steroids such as 5 beta-androstane-17 beta-ol-3-one. Molecular modeling suggests that the differences between MmADH alpha and EqADH are a result of the substitution of Ala for Phe93 and Thr for Ser48. MmADH alpha binds NAD+ most rapidly when a group with a pK of 7.4 is unprotonated, implicating His51 in this reaction. The G47R substitution decreased the dissociation constants for NAD+ and NADH and turnover numbers only about 2-fold, whereas the G47H substitution increased dissociation constants 7-14-fold and turnover numbers 4-fold. A basic residue at position 47 is not crucial for activity, as multiple interactions determine coenzyme affinity.