Glyceraldehyde-3-phosphate dehydrogenase

Conflicting experimental evidence of the pathway of catalysis for the enzyme from rabbit, pig and lobster muscle tissues is reviewed. Transient kinetic studies with the enzyme from rabbit muscle are presented. The results are shown to be consistent with the double-displacement mechanism of catalysis originally proposed by Segal & Boyer (1953). The rate constant for combination of the aldehyde form of the substrate with the NAD+ complex of the enzyme is about 3 X 10(7) M-1 S-1, and for all four subunits of the molecule the rate constant for hydride transfer in the ternary complex formed is greater than 10(3) S-1, consistent with their simultaneous participation in catalysis. Recent steady-state kinetic studies with the rabbit muscle enzyme, in contrast to earlier studies, also provide evidence to support the Segal-Boyer pathway if the kinetic effects of the negative cooperativity of NAD+ binding are taken into account. Experimental data for the binding of NAD+ to the enzyme from muscles and from Bacillus stearothermophilus, and their interpretations, are also briefly reviewed. The information currently available from X-ray crystallography regarding the structures of holoenzyme and apoenzyme from B. stearothermophilus and lobster muscle is outlined.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

Inhibition of glycerol-3-phosphate acyltransferase by analogs of glycerol-3-phosphate.

Analogs of glycerol-3-phosphate were tested as substrates or inhibitors of the glycerol-3-phosphate acyltransferases of mitochondria and microsomes. (rac)-3,4-Dihydroxybutyl-1-phosphonate, (rac)-glyceraldehyde 3-phosphate, (rac)-3-hydroxy-4-oxobutyl-1-phosphonate, (1S,3S)-1,3,4-trihydroxybutyl-1-phosphonate, and (1R,3S)-1,3,4 trihydroxybutyl-1-phosphonate were competitive inhibitors of both mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase activity. An isosteric analog of dihydroxyacetone phosphate, 4-hydroxy-3-oxobutyl-1-phosphonate, was a much stronger competitive inhibitor of the microsomal than the mitochondrial enzyme. Phenethyl alcohol was a noncompetitive inhibitor of both the microsomal and the mitochondrial acyltransferases. The product of the mitochondrial acyltransferase reaction with (rac)-3,4-dihydroxybutyl-1- phosphonate was almost exclusively (rac)-4-palmitoyloxy-3-hydroxybutyl-1-phosphonate. The microsomal acylation reaction generated both the monoacyl product and (S)-3,4-dipalmitoyloxybutyl-1-phosphonate. The apparent Km for (S)-3,4-dihydroxybutyl-1-phosphonate was 2.50 and 1.38 mM for the mitochondrial and microsomal enzymes, respectively.     

d-Sedoheptulose-7-phosphate: d-Glyceraldehyde-3-phosphate Glycolaldehydetransferase and d-Ribulose-5-phosphate 3-Epimerase Mutants of a Bacillus Species

Determination of enzyme activities on the non-oxidative section of the pentose phosphate pathway in d-ribose-forming mutants of a Bacillus species revealed that two strains, which were isolated as shikimic acid-requiring mutants, lacked d-sedoheptulose-7-phosphate: d-glyceraldehyde glycolaldehydetransferase (EC 2.2.1.1) and one strain, which was isolated as d-gluconate-non-utilizing mutant, lacked d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1). These three strains were also found to have a kind of pleiotropic property, hardly growing on d-glucose.     

Properties of 3-hydroxypropionaldehyde 3-phosphate.

Several modifications to the synthesis of the diethyl acetal of 3-hydroxypropionaldehyde-3-P (HPAP) are described. HPAP is liberated from its acetal by treatment with Dowex 50-H⁺ at 40 °C for 4 min, and longer time or higher temperature lower yields. Breakdown of the dianion of HPAP (pK 6.7) is self-catalyzed, with the phosphate acting as a general base to remove a proton from carbon 2 and allow elimination of phosphate to give acrolein. Monoanion breakdown is at least 400-fold slower. At 25 °C the dianion breaks down with k = 0.025 min^?1, and the activation energy for the process is 24 kcal/mol. Buffers have little effect on breakdown of HPAP, except for those containing primary or secondary amines. Thus morpholine enhances breakdown by forming a Schiff's base with a positively charged nitrogen, and Tris inhibits breakdown by forming one with an uncharged nitrogen. The aldehyde group of HPAP is 60% hydrated in water.     

Reaction of triosephosphate isomerase with L-glyceraldehyde 3-phosphate and triose 1,2-enediol 3-phosphate

Triosephosphate isomerase catalyzes the isomerization and/or racemization reactions of L-glyceraldehyde 3-phosphate (LGAP), the enantiomer of the physiological substrate. The reaction is inhibited by the active site directed reagent glycidol phosphate. The amount of protonation product formation catalyzed by a fixed enzyme concentration is nearly independent of increasing steady-state concentrations of triose 1,2-enediol 3-phosphate caused by buffer catalysis of LGAP deprotonation. Therefore, enzymatic protonation of the enediol or enediolate, which could account for the observed enzymatic catalysis of LGAP isomerization and/or racemization, is at best a minor reaction. Instead LGAP reacts directly at the enzyme active site. Triosephosphate isomerase catalysis of the protonation of triose 1,2-enediol 3-phosphate was expected because of the strong evidence supporting an enediol reaction intermediate for the overall reaction catalyzed by isomerase. The most reasonable explanation for the failure to observe enzymatic protonation is that in solution the enediol undergoes beta elimination of phosphate (t 1/2 is estimated to be 10(-6) s) faster than it can diffuse to and form a complex with isomerase.     

Simple Procedures for the Preparation of d-Ribose-5-phosphate Ketol-Isomerase,d-Ribulose-5-phosphate 3-Epimerase and d-Sedoheptulose-7-phosphate: d-Glyceraldehyde-3-phosphate Glycolaldehydetransferase

d-Ribose-5-phophate ketol-isomerase (EC 5.3.1,6), d-ribuIose-5-phosphate 3-epimerase (EC 5.1.3.1) and d-sedoheptulose-7-phosphate: d-gIyceraldehyde-3-phosphate glycolaldehyde-transferase (EC 2.2.1,1) have been partially purified. d-Ribose-5-phosphate ketol-isomerase was purified from spinach by column chromatography with DEAE-cellulose and DEAE-Sephadex A-50; d-ribulose-5-phosphate 3-epimerase was purified from baker’s yeast by column chromatography with DEAE-cellulose; and d-sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase was purified from a Bacillus species No. 102 mutant G3–46–22–6 by column chromatography with DEAE-cellulose. The preparations were used for the determination of the activities of these enzymes in the parent and d-ribose-forming mutants of a Bacillus species.     

Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.     

Identification of sorbitol 3-phosphate and fructose 3-phosphate in normal and diabetic human erythrocytes

Using 31P NMR spectroscopy, we have identified sorbitol 3-phosphate and fructose 3-phosphate in normal human erythrocytes wherein their concentrations are estimated to be 13 mumol/liter cells. Incubation of hemolysates with sorbitol, fructose and ATP suggest that both sorbitol and fructose are phosphorylated separately and directly at the 3-hydroxyl position suggesting the presence in these cells of a novel and specific kinase(s). In addition to sorbitol 3-phosphate and fructose 3-phosphate which were previously identified in the mammalian lens and sciatic nerve, erythrocytes have two extra metabolites resonating at 6.7 and 6.8 ppm in the 31P NMR spectrum. Although not identified in this study, the unusual chemical shifts of these compounds, their low pKa values and the fact that they appear as doublet in proton-coupled 31P NMR spectra, suggest that these phosphomonoesters belong to the same class of metabolites as sorbitol 3-phosphate and fructose 3-phosphate. Preliminary studies of erythrocytes from an unselected group of diabetic subjects showed an overall increase in the concentration of all four metabolites, although an overlap with normal values was noted.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

Glycerol-3-phosphate and systemic immunity

Glycerol-3-phosphate (G3P), a conserved three-carbon sugar, is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis. G3P can be derived via the glycerol kinase-mediated phosphorylation of glycerol or G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate. Previously, we showed G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in GLY1 encoded G3Pdh accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Recently, we showed that G3P is also a potent inducer of systemic acquired resistance (SAR) in plants. SAR is initiated after a localized infection and confers whole-plant immunity to secondary infections. SAR involves generation of a signal at the site of primary infection, which travels throughout the plants and alerts the un-infected distal portions of the plant against secondary infections. Plants unable to synthesize G3P are defective in SAR and exogenous G3P complements this defect. Exogenous G3P also induces SAR in the absence of a primary pathogen. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues. Together, these observations suggest that the cooperative interaction of DIR1 and G3P mediates the induction of SAR in plants.Glycerol-3-phosphate (G3P) is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis.^1,2 G3P levels in the plant are regulated by enzymes directly/indirectly involved in G3P biosynthesis, as well as those involved in G3P catabolism. G3P is synthesized via the glycerol kinase (GK)-mediated phosphorylation of glycerol,³ or the G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP)⁴ (Fig. 1). DHAP is derived from glycolysis via triosephosphate isomerase activity on glyceraldehyde-3-phosphate, or from the conversion of glycerol to dihydroxacetone (DHA) by glycerol dehydrogenase (Glydh) followed by phosphorylation of DHA to DHAP by DHA kinase (DHAK). G3P is catabolized either upon its conversion to glycerol by glycerol-3-phoshatase (GPP) or its utilization in glycerolipid/triacylglycerol biosynthesis. In Arabidopsis, the total G3P pool is derived from the activities of five G3Pdh isoforms and one GK isoform present in three cellular locations^5-9; GK and two of the G3Pdh isoforms are present in the cytoplasm, two other G3Pdh isoforms localize to plastids, and one to the mitochondria. One of the plastid localized G3Pdh isoforms, designated GLY1, was previously shown to be required for glycerolipid biosynthesis; a mutation in GLY1 compromised lipids synthesized via the plastidal pathway of lipid biosynthesis. The fact that exogenous application of glycerol to gly1 plants normalizes plastidal lipid levels¹⁰ and that GLY1 encodes a G3Pdh⁴ suggests that the G3P pool generated via the GLY1 catalyzed reaction is required for the biosynthesis of plastidal lipids. Intriguingly, unlike GLY1, neither the chloroplastic, nor the two cytosolic isoforms of G3Pdh, contribute to plastidal and/or extraplastidal lipid biosynthesis.⁹Open in a separate windowFigure 1.A condensed scheme of glycerol-3-phosphate metabolism in plants. Glycerol is phosphorylated to glycerol-3-phosphate (G3P) by glycerol kinase (GK; GLI1). G3P can also be generated by G3P dehydrogenase (G3Pdh) via the reduction of dihydroxyacetone phosphate (DHAP). DHAP is derived from glycolysis via triosephosphate isomerase (TPI) activity on glyceraldehyde-3-phosphate (Gld-3-P), or from the conversion of glycerol to dihydroxacetone (DHA) by glycerol dehydrogenase (Glydh) followed by phosphorylation of DHA to DHAP by DHA kinase (DHAK). G3Pdh isoforms are present in both the cytosol and the plastids (represented by the oval). GLY1 is one of the two plastidial G3Pdh isoforms that plays an important role in plastidial glycerolipid biosynthesis. In the plastids, G3P is acylated with oleic acid (18:1) by the ACT1-encoded G3P acyltransferase. This ACT1-utilized 18:1 is derived from the stearoyl-acyl carrier protein (ACP)-desaturase (SACPD)-catalyzed desaturation of stearic acid (18:0). The 18:1-ACP generated by SACPD either enters the prokaryotic lipid biosynthetic pathway through acylation of G3P or is exported out (dotted line) of the plastids as a coenzyme A (CoA)-thioester to enter the eukaryotic lipid biosynthetic pathway. Membranous fatty acid desaturases (FAD) catalyze desaturation of FAs present on membranous glycerolipids. Other abbreviations used are: GL, glycerolipid; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; Lyso-PA, acyl-G3P; PA, phosphatidic acid; PG, phosphatidylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SL, sulfolipid; DAG, diacylglycerol.For glycerolipid biosynthesis, G3P is first acylated with the fatty acid (FA) oleic acid (18:1), to form lyso-phosphatidic acid (lyso-PA) via the activity of the soluble G3P acyltransferase (GPAT) encoded by the ACT1 gene in Arabidopsis¹¹ (Fig. 1). 18:1 in turn is derived from the saturated FA, stearic acid (18:0), via the activity of soluble stearoyl-acyl carrier protein desaturases (SACPD),¹² which introduce a single cis double bond in 18:0. The 18:1 generated via this reaction is either exported out of the plastids or acylated at the sn-1 position of G3P. Previously, we have shown that 18:1 levels are important regulators of plant defense signaling. In Arabidopsis, 18:1 is synthesized via the SSI2/FAB2-encoded SACPD,¹² which uses 18:0 as a substrate. A mutation in SSI2 results in the accumulation of 18:0 and a reduction in 18:1 levels. The mutant plants show stunting, spontaneous lesion formation, constitutive PR gene expression, and enhanced resistance to bacterial and oomycete pathogens.^4,12-17 Characterization of ssi2 suppressor mutants has shown that the altered defense-related phenotypes are the result of the reduction in the levels of the unsaturated FA, 18:1, which causes induction of several resistance (R) genes.^4,14,18,19 Restoration of 18:1 levels, via mutations in ACT1,¹⁴ GLY1⁴ or ACP4,¹⁸ normalizes R gene expression in ssi2 plants. The low 18:1-mediated induction of R gene expression and the associated defense signaling can also be suppressed by simultaneous mutations in EDS1 and the genes governing salicylic acid (SA) biosynthesis (SID2, EDS5).¹⁹ Furthermore, the functional redundancy between EDS1 and SA likely masks the requirement for EDS1 by several coiled coil (CC)- nucleotide binding site (NBS)- leucine rich repeat (LRR) proteins,¹⁹ previously thought to function independent of EDS1.²⁰ Thus, the reliance on EDS1 for signaling mediated by CC-NBS-LRR proteins becomes evident only in the absence of SA.The plastidal 18:1 levels are also regulated via the chloroplastic G3P pool and vice-versa. However, 18:1 and G3P appear to function distinctly in defense signaling. For example, G3P levels are important for basal defense against the hemibiotrophic fungus, Colletotrichum higginsianum.^21,22 Genetic mutations affecting G3P synthesis in Arabidopsis enhance susceptibility to C. higginsianum. Conversely, plants accumulating increased G3P show enhanced resistance. More recently, we demonstrated roles for G3P in R-mediated defense leading to systemic acquired resistance (SAR).⁹ R-mediated defense against the avirulent bacterial pathogen P. syringae is associated with a rapid increase in G3P levels; G3P levels peak within 6 h of inoculation with avirulent bacteria (avrRpt2), in resistant plants expressing the R gene RPS2. Strikingly, accumulation of G3P, in the infected and systemic tissues, precedes the accumulation of other metabolites known to be essential for SAR; SA,^23,24 jasmonic acid (JA)²⁵ and azelaic acid (AA)²⁶ accumulated at least 24 h post pathogen inoculation. Furthermore, mutants defective in G3P synthesis are compromised in SAR but accumulated normal levels of SA, AA, and JA. Compromised SAR in G3P deficient mutants was restored by exogenous application of G3P, thus arguing a role for G3P in SAR. This was further supported by the fact that exogenous G3P induced SAR in the absence of the primary pathogen in both Arabidopsis and soybean.⁹ That fact that G3P is a conserved metabolite common to prokaryotes, plants, and humans further corroborates the conserved nature of SAR signaling. Interestingly, although exogenous G3P did not induce SA biosynthesis, SAR conferred by exogenous G3P was dependent on SA. These results suggest that the onset and/or establishment of SAR likely requires basal, but not induced levels of SA, in the distal tissues. It is possible that the relatively small increase in SA observed in the systemic tissues during SAR is an indirect response that contributes to generalized resistance, rather than SAR itself. Interestingly, both G3P conferred SAR, and the systemic movement of G3P were dependent on the lipid transfer protein, DIR1, a well-known positive regulator of SAR.²⁷ Conversely, systemic movement of DIR1 required G3P. These findings did not correlate with the fact that G3P is cytosolic while DIR1 was a predicted apoplastic protein. To resolve this issue, we studied the localization of DIR1, and found that it is in fact a symplastic protein. The symplastic location of DIR1 was further corroborated when GFP fused to the signal peptide from DIR1 localized to the endoplasmic reticulum, rather than the typical cytoplasmic and nuclear location of GFP (Fig. 2). These results suggested that the symplastic movement of DIR1 is likely critical for SAR, and supported the facts that G3P and DIR1 are interdependent for translocation to systemic tissues. However, these findings could not explain how a lipid transfer-like protein might associate with the phosphorylated sugar G3P, to move systemically. Analysis of G3P in the leaf extracts showed that it was derivatized into an unknown compound before/during translocation. It is likely that the G3P derivative has a lipid moiety via which it associates with DIR1 for transfer. In summary, we showed that DIR1 together with a G3P-derived compound are sufficient for the induction of SAR in wild type plants. Our findings provide strong evidence in support of a direct defense-signaling role for G3P and warranty further analysis of its metabolic pathway(s) for their role(s) in various modes of plant defense.Open in a separate windowFigure 2.Confocal micrograph showing localization of GFP fused to DIR1 transit peptide (TP) or GFP alone in Nicotiana benthamiana plants expressing RFP-tagged nuclear histone protein H2B. Arrow indicates nucleus, arrowhead indicates endoplasmic reticulum.     

Iodination of glyceraldehyde 3-phosphate dehydrogenase

1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[(125)I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed.     

sn-Glycerol-3-phosphate acyltransferases in plants

sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA). LPA is an important intermediate for the formation of different types of acyl-lipids, such as extracellular lipid polyesters, storage and membrane lipids. Three types of GPAT have been found in plants, localizing to the plastid, endoplasmic reticulum, and mitochondria. These GPATs are involved in several lipid biosynthetic pathways and play important biological roles in plant development. In the present review, we will focus on the recent progress in studying the physiological functions of GPATs and their metabolic roles in glycerolipid biosynthesis.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.