3'-Arylazido-8-azido ATP--a cross-linking photoaffinity label for ATP binding proteins

The synthesis of 3′-O-{3-[N-(4-azido-2-nitrophenyl) amino] propionyl} 8-azido-adenosine 5′-triphosphate—a 3′-arylazido-8-azido ATP—is described. The ATP derivative is characterized by thin layer chromatography, infrared spectroscopy, and optical spectroscopy. Its photolysis upon irradiation with uv light and its stability in dependence on pH are tested. Its two photolabile azido groups allow the use of this ATP analog as a photoaffinity label for cross-linking the subunits of ATP binding proteins.     

Photoaffinity labelling of a thromboxane A2/prostaglandin H2 antagonist binding site in human platelets

The diazonium salt of 9,11-dimethylmethano-11,12-methano-16-(4-aminophenoxy)13,14- dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 (PTA-POA) was synthesized and used as a photoaffinity ligand for the putative human platelet TXA2/PGH2 receptor. Incubation of human platelet membranes with the diazonium salt of PTA-POA followed by photolysis at 290 nm(hv) resulted in a 40% decrease in the specific binding of [125I]PTA-OH as measured in the radioligand binding assay. Co-incubation with a TXA2/PGH2 agonist followed by photolysis resulted in no decrease in specific binding. Incubation of the diazonium salt of PTA-POA with solubilized platelet membranes without photolysis followed by Scatchard analysis resulted in no change in the Kd for [125I]PTA-OH (38 nM) and the preparation which was incubated with the diazonium salt (42 nM). However, the Bmax for [125I]PTA-OH binding was reduced from 2.4 pmole/mg protein for control to 1.4 pmole/mg protein. These studies show that the diazonium salt of PTA-POA may be a useful photoaffinity ligand for human platelet TXA2/PGH2 receptors.     

Photoaffinity labeling of specific muscarinic antagonist binding sites of brain: I. Preliminary studies using two p-azidophenylacetate esters of tropine

Possible photoaffinity probes for muscarinic acetylcholine receptors have been explored for the first time: Specific [³H]-quinuclidinyl benzylate binding sites of several fractions from rat brain can be irreversibly inactivated by photoaffinity labeling with two p-azidophenylacetate esters of tropine. Inactivation of these sites depends on formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes; it is dependent on ligand concentration and length of photolysis. Atropine and oxotremorine, but not d-tubocurarine, afford protection against photoinactivation.These findings suggest the utility of these and related azido derivatives as potent, selective photoaffinity ligands directed against binding sites for muscarinic antagonists.     

Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.     

Photoaffinity labelling of isopenicillin N synthetase.

The alpha-aminoadipoyl group of the natural substrate of isopenicillin N synthetase (IPNS), L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), has been replaced by a diazirinyl-containing group, which can be photoactivated. This has allowed investigation of the substrate binding site of IPNS by photoaffinity labelling. Laser flash photolysis of this analogue, [3H]DCV, in the presence of IPNS leads to the incorporation of radioactivity into the enzyme. Tryptic digestion of this labelled enzyme, followed by separation and sequencing of the resultant fragments, identified two labelled regions of the protein. These are the fragments Asp-40 to Arg-78 and Thr-237 to Gly-256.     

p-Toluenesulfonyldiazoacetates: Reagents for photoaffinity labeling

p-Toluenesulfonyldiazoacetyl chloride and p-nitrophenyl p-toluenesulfonyldiazoacetate have been prepared and offer potential advantages as reagents for photoaffinity labeling. (i) The extinction coefficient for the sulfonyldiazo compounds at 370 nm is about 10 times that for the long wavelength absorption of other diazoesters; this absorption permits reasonably rapid photolysis in the presence of compounds that are destroyed by short wavelength uv radiation. (ii) The two derivatives named above are stable thermally; furthermore, since sulfonyldiazoesters are stable to acid and to weak base, photoaffinity labeling can be conducted over a wide range of pH. (iii) Photolysis of ordinary (i.e., oxygen) esters of sulfonyldiazo compounds in methanol or cyclohexane leads to insertion into the solvent to the exclusion of Wolff rearrangement; photolysis of thioesters at 350 nm in methanol gives about 25% insertion into solvent, accompanied by about 75% Wolff rearrangement; in contrast, photolysis of most thioesters of diazo derivatives leads exclusively to Wolff rearrangement     

Photoreactive derivative of Kunitz's soybean trypsin inhibitor. Preparation by selective modification of a tryptophan residue and formation of a covalent complex of the modified inhibitor with trypsin

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1-114 and 115-181. Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [3H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.     

Leukotriene B4 photoaffinity probes: design, synthesis and evaluation of new arylazide-1,3-disubstituted cyclohexanes

The synthesis and the binding affinities of new leukotriene B4 receptor photoaffinity probes, where a 1,3-disubstituted cyclohexane ring replaces the conjugated delta6,7 and delta8,9 double bonds of the natural eicosanoid, are described. One enantiomeric compound, 4b alpha, is specifically cross-linked upon photolysis to the recombinant leukotriene B4 receptor from human origin (h-BLTR) solubilized in a micellar medium. This probe appears as a good candidate for identifying the ligand binding site of this receptor.     

A photoaffinity derivative of colchicine: 6'-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine. Photolabeling and location of the colchicine-binding site on the alpha-subunit of tubulin

A photoaffinity analog of colchicine, 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine, was synthesized by reacting deacetylcolchicine or [3H]deacetylcochicine with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Homogeneity of the photoaffinity analog was established by thin-layer chromatography and high-pressure liquid chromatography. The structure of the photoaffinity analog was determined by 1H and 13C NMR, infrared and ultraviolet-visible spectroscopies, and elemental analysis. Binding of 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine to bovine renal tubulin was measured by competition with [3H]colchicine. The value of the apparent Ki for the photoaffinity analog was 0.28 microM in the concentration range of 0.8-1.2 microM of the analog. A value of 0.50 microM for the apparent Kd was measured by the direct binding of the tritiated photoaffinity analog to tubulin. The analog is slightly more potent an inhibitor of microtubule formation than colchicine. The photoaffinity analog reacted with renal tubulin upon irradiation with a mercury lamp equipped with a 420-nm cutoff filter. Spectral and radiochemical analyses of the tubulin after photolysis and dialysis have demonstrated a stoichiometric incorporation of the photoaffinity analog in the alpha-subunit of the tubulin. Covalent labeling of tubulin with the photoaffinity analog decreases the extent of [3H]colchicine binding by more than 90%.     

Interaction of glycogen phosphorylase with 8-azidoadenosine 5'-monophosphate, a photoaffinity analog of AMP

The ability of 8-azidoadenosine 5'-monophosphate (N3AMP) to act as a photoaffinity label for the AMP binding site on glycogen phosphorylase (EC 2.4.1.1) was tested. 8-Azidoadenosine 5'-monophosphate can replace AMP as an allosteric modifier of both phosphorylases a and b; the pH optimum and the extent of activation are comparable to that observed with AMP. 8-Azidoadenosine 5'-monophosphate resembles the natural activator in having a higher affinity for phosphorylase a. The effects of 8-azidoadenosine 5'-monophosphate and AMP on phosphorylase b are additive when each is present at a concentration which gives less than 50% activation. Increasing the concentration of the substrate, glucose 1-phosphate, decreases the apparent activation constant (Ka) for the interaction of 8-azidoadenosine 5'-monophosphate with phosphorylase b. Glucose 6-phosphate is an inhibitor of phosphorylase b with either AMP or 8-azidoadenosine 5'-monophosphate. In the presence of ultraviolet light, 8-azidoadenosine 5'-monophosphate is irreversibly incorporated into phosphorylase a; incorporation at the allosteric site can be reduced if AMP is added prior to irradiation. Under the conditions used in the photolysis experiments, 3--5% of the available AMP sites were labeled with 8-azidoadenosine 5'-monophosphate. The data indicate the potential usefulness of 8-azidoadenosine 5'-monophosphate as a probe for the AMP site on phosphorylase.     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

Evaluation of 3-azidiamantane as photoaffinity probe of cytochrome P450.

3-azidiamantane (DIA-N2) has been shown to be a photolabile carbene-generating probe interacting specifically with cytochrome P450 (P450) active centre. To evaluate the modification of P450 by the probe, radiolabelled [9-3H]-3-azidiamantane was prepared by reductive dehalogenation of its precursor, 3-oxo-9-bromodiamantane ethylene ketal. The synthesis was optimized as the proper precursor and reaction conditions were concerned to produce 96% pure product (overall yield 59%). An incorporation efficacy of the probe photoactivated at 366 nm was examined with two different proteins, BSA and rat phenobarbital-inducible P450 2B1, both having hydrophobic binding sites. Under photolysis the photoaffinity probe generated short-lived (> 90%) intermediates binding immediately to the protein. The yield of photoactivated DIA-N2 incorporation was 12% and 11% for BSA and P450, respectively. The presence of reduced glutathione, a scavenger of reactive intermediates, did not affect the probe incorporation markedly. On the other hand, scavengers entering the P450 active centre, methanol and dithiothreitol, reduced the protein labelling by 36% and 42%, respectively. Similarly, at DIA-N2, aminopyrine (substrates), and metyrapone (inhibitor) 50 times molar excess over the probe, prevented its binding by about 40%. In addition, when photoaffinity labelling was carried out with microsomal preparation, the substrate with a high affinity for the P450 2B1, diamantane, (at 20 times molar excess to the probe) caused 47% inhibition of the P450 covalent labelling. These results, suggesting a high specificity of the probe binding, show that it can be applied as a photoaffinity probe for cytochrome P450 2B1 active centre studies.     

Inactivation of trypsin and chymotrypsin with a photosensitive probe.

The photosensitive inactivation of trypsin and chymotrypsin by 4-fluoro-3-nitrophenyl azide (FNPA) is described. A dark inhibition was observed at elevated probe concentrations, and was reversible. The enzymes were stable to photolysis in the absence of probe. Photolytic inactivation of trypsin and chymotrypsin with FNPA was found to be irreversible, and occurs in minutes at concentrations of FNPA where dark inhibition is negligible. The photoprobe was equally effective at pH 3 or pH 8. Nonspecific inactivation appears to be low, as evidenced by the stability of glucose oxidase and peroxidase to photolysis with FNPA.     

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.     

Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.     

Production of frameshift mutations in salmonella by a light sensitive azide analog of ethidium

Frameshift mutations have been produced in specific repair-negative Salmonella tester strains by photoaffinity labeling technique using ethidium azide. Reversions requiring a +1 addition or a ?2 deletion were especially sensitive. Mutagenesis was reduced by the simultaneous addition of non-mutagenic ethidium bromide, and was prevented by photolysis of the azide prior to culture addition. Identical tester strains active in DNA excision repair were not mutagenized by the azide. These results are consistent with the interpretation that photolysis of the bound ethidium analog converts the drug from its noncovalent mode of binding (presumably intercalation) to a covalent complex with consequent production of frameshift mutations. Such photoaffinity labeling by drugs which bind to DNA not only confirms the importance of covalent drug attachment for frameshift mutagenesis, but also provides powerful techniques for studying the molecular details of a variety of genetic mechanisms.     

Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase

The adenine binding domain of the ADP site within human glutamate dehydrogenase (GDH) was identified by cassette mutagenesis at the Tyr187 position. The wild type GDH was activated 3-fold by ADP at a concentration of 1 mM at pH 8.0, whereas no significant activation by ADP was observed with the Tyr187 mutant GDH regardless of the size, hydrophobicity, and ionization of the side chains. Studies of the steady-state velocity of the mutant enzymes revealed essentially unchanged apparent K(m) values for 2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective apparent V(max) values. The binding of ADP to the wild type or mutant GDH was further examined by photoaffinity labeling with [alpha-(32)P]8-azidoadenosine 5'-diphosphate (8N(3)ADP). 8N(3)ADP, without photolysis, mimicked the stimulatory properties of ADP on GDH activity. Saturation of photoinsertion with 8N(3)ADP occurred with apparent K(d) values near 25 microM for the wild type GDH, and the photoinsertion of [alpha-(32)P]8N(3)ADP was decreased best by ADP in comparison to other nucleotides. Unlike the wild type GDH, essentially no photoinsertion was detected for the Tyr187 mutant GDH in the presence or absence of 1 mM ADP. For the wild type GDH, photolabel-containing peptide generated by tryptic digestion was identified in the region containing the sequence EMSWIADTYASTIG, and the photolabeling of this peptide was prevented >95% by the presence of 1 mM ADP during photolysis, whereas no such a peptide was detected for the Tyr187 mutant GDH in the presence or absence of ADP. These results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide is within the ADP binding domain of the human GDH and that Tyr187 is responsible for the efficient base binding of ADP to human GDH.     

Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan

The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 microM). Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.     

7-Azidoactinomycin D: a novel probe for examining actinomycin D-DNA interactions

The technique of photoaffinity labeling is applied to the actinomycin D system to provide a novel probe for the examination of the interactions of actinomycin D with nucleic acids. The capacity for covalent attachment of actinomycin D will aid greatly in the study of target-site specificities and the correlations of biological effects with biophysical DNA interactions. Through chemical modification of the parent actinomycin D molecule with a photoreactive azido substituent, a functional analog of the parent actinomycin D is generated having equilibrium binding properties identical to those of the parent molecule yet with the capacity to form a covalent attachment to DNA upon photolysis. The results presented here describe the noncovalent interactions of this photoreactive probe to DNA (absence of light) and compares the binding properties observed to those of the parent actinomycin D and 7-aminoactinomycin D analog. These studies demonstrate that the DNA binding properties (i.e. binding affinity, binding site size, and sequence specificity) retained by the 7-azidoactinomycin D, thus providing a suitable probe for examining actinomycin D-DNA interactions.     

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.