Aspects of the chemistry of d-glyceraldehyde 3-phosphate dehydrogenase

Crystalline d-glyceraldehyde 3-phosphate dehydrogenase from lobster tail contains 4 moles of NAD(+) bound and reacts specifically with 4 moles of iodoacetic acid/mole of tetramer. The essential thiol group of d-glyceraldehyde 3-phosphate dehydrogenase appears to react with iodoacetic acid with a rate constant for the overall process that is independent of the extent of carboxymethylation. The d-glyceraldehyde 3-phosphate dehydrogenase-NAD(+) absorption band has a variable molar extinction coefficient in the presence of phosphate that may be correlated with a proton dissociation of pK 6.86. The binding of NAD(+) to d-glyceraldehyde 3-phosphate dehydrogenase weakens as alkylating agents react with the enzyme, and NAD(+) promotes the reactivity of the essential thiol group. It is suggested that, on binding to d-glyceraldehyde 3-phosphate dehydrogenase, NAD(+) lowers the pK of the essential thiol group, resulting in a catalytic role of NAD(+) in the reaction catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. If this theory is correct, then it is likely that a proton will be liberated during the phosphorolysis of the acyl-enzyme rather than in the redox step.     

An examination of the role of arginine residues in the functioning of D-glyceraldehyde-3-phosphate dehydrogenase

Chemical modification of one arginine residue per subunit of tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) molecule results in a 85-95% loss of its activity (Nagradova and Asryants (1975) Biochim. Biophys. Acta 386, 365-368; Nagradova, N.K., Asryants, R.A., Benkevich, N.V. and Safronova, M.I. (1976) FEBS Lett. 69, 246-248). Transient kinetic experiments performed in the present work with modified rabbit muscle and Baker's yeast enzymes showed that the first-order rate constant of acyl-enzyme.NADH formation was diminished 30-fold with the rabbit muscle enzyme and 60-fold with the Baker's yeast enzyme. Modification of arginine residues was shown also to affect the second step of the catalytic reaction, the phosphorolysis of the acyl-enzyme (the second-order rate constant of phosphorolysis decreased 9-fold in the case of the rabbit muscle enzyme and 40-fold in the case of the Baker's yeast enzyme). The native and modified enzymes exhibited similar inhibitory constant values with respect to NADH, suggesting no contribution of arginine residues to the acyl-enzyme.NADH complex destabilization. By and large, the experimental data are consistent with the hypothetical scheme proposed on the basis of X-ray crystallography studies to describe a participation of Arg-231 in the catalytic mechanism of D-glyceraldehyde-3-phosphate dehydrogenase (Grau (1982) in the Pyridine Nucleotide Coenzymes, p. 135-187).     

Kinetic studies of oxidized nicotinamide–adenine dinucleotide-facilitated reactions of d-glyceraldehyde 3-phosphate dehydrogenase

The kinetics of the acylation of d-glyceraldehyde 3-phosphate dehydrogenase from pig muscle by 1,3-diphosphoglycerate in the presence of NAD(+) has been analysed by using the relaxation temperature-jump method. At pH7.2 and 8 degrees C the rate of acylation of the NAD(+)-bound (or holo-) enzyme was 3.3x10(5)m(-1).s(-1) and the rate of phosphorolysis, the reverse reaction, was 7.5x10(3)m(-1).s(-1). After a temperature-jump perturbation the equilibrium of NAD(+) binding to the acyl-enzyme was re-established more rapidly than that of the acylation. The rate of phosphorolysis of the apoacylenzyme from sturgeon muscle and of aldehyde release from the d-glyceraldehyde 3-phosphate-apoenzyme complex were 相似文献   

Interaction of inorganic vanadate with glucose-6-phosphate dehydrogenase. Nonenzymic formation of glucose 6-vanadate

Inorganic vanadate (Vi) activates catalysis by glucose-6-phosphate dehydrogenase of the oxidation of glucose by NADP+. As the concentration of Glu-6-P dehydrogenase is increased, the rate of the vanadate-activated glucose oxidation becomes less sensitive to increases in enzyme concentration. The rate of glucose oxidation in the absence of Vi increases linearly with Glu-6-P dehydrogenase concentration. These results are interpreted in terms of nonenzymic formation of glucose 6-vanadate. At high enzyme concentration, vanadate ester formation becomes partially rate-limiting, and extrapolation to infinite Glu-6-P dehydrogenase concentration allows determination of the second order rate constant for formation of the ester. In separate experiments designed to test the proposed mechanism, it was found that Vi, at concentrations at which it strongly activates catalysis by Glu-6-P dehydrogenase of glucose oxidation, has no effect on the rates of oxidation of glucose 6-phosphate or 6-deoxyglucose catalyzed by Glu-6-P dehydrogenase. Sulfate, which is known to activate glucose oxidation and to inhibit glucose 6-phosphate oxidation, strongly activates 6-deoxyglucose oxidation. These experiments show that the 6-hydroxyl group of glucose is essential for the observed activation by Vi and are also consistent with the formation of glucose 6-vanadate. Also, the rate of the sulfate-activated glucose oxidation increases linearly with Glu-6-P dehydrogenase concentration. These results are consistent with the proposed mechanism for sulfate activation which involves sulfate binding to the enzyme (Anderson, W. B., Horne, R. N., and Nordlie, R. C. (1968) Biochemistry 7, 3997-4004). The second order rate constant calculated for formation of glucose 6-vanadate at pH 7.0 is 2.4 M-1 s-1. The corresponding values for glucose 6-phosphate and glucose 6-arsenate formation are approximately 9 X 10(-11) M-1 s-1 and 6.3 X 10(-6) M-1 s-1 (Lagunas, R. (1980) Arch. Biochem. Biophys. 205, 67-75).     

Kinetic evidence for interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase.

The possibility of interaction between purified rabbit muscle aldolase and D-glyceraldehyde-3-phosphate dehydrogenase was studied by rapid kinetic methods, by analyzing the kinetics of the consecutive reaction catalyzed by the coupled enzyme system. The Km of the intermediary product, glyceraldehyde 3-phosphate, produced by aldolase was determined in the coupled reaction for glyceraldehyde-3-phosphate dehydrogenase. Its value corresponds to that of the aldehyde (active) form of glyceraldehyde 3-phosphate, although in the given conditions the aldehyde leads to diol interconversion is faster than the enzymic reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. We suggest that above a certain concentration of the enzymes the glyceraldehyde 3-phosphate produced by aldolase gets direct access to glyceraldehyde-3-phosphate dehydrogenase without participating in the aldehyde leads to diol interconversion which otherwise would occur if the substrate were to mix with the bulk medium.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate

The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate, D-glyceraldehyde 3-phosphate (D-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys(149), was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals of the corresponding binary complexes (enzyme.NAD) in a solution containing G3P. The structures of the two binary and the two ternary complexes are presented. The D-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form, its C-3 phosphate group being positioned in the P(s) site and not in the P(i) site. Its C-1 carbonyl oxygen points toward the essential His(176), which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that the structures reported here are representative of a productive enzyme.NAD.D-G3P complex in the ground state (Michaelis complex).     

Modification of glyceraldehyde 3-phosphate dehydrogenase activity by adsorption on phospholipid vesicles.

1. The adsorption of [14C]carboxymethylated glyceraldehyde 3-phosphate dehydrogenase to negatively charged liposomes of phsphatidic acid/phosphatidylcholine (3:7, w/w) was investigated. The apparent association constant at I/2 = 60, pH 7.6, was 0.4 X 10(6)M-1. Adsorption decreased as ionic strength and pH were increased. 2. In the presence of negatively charged liposomes, the Km value for glyceraldehyde 3-phosphate of glyceraldehyde 3-phosphate dehydrogenase was increased and Vmax. decreased. In the presence of positively charged liposomes, the Km value for glyceraldehyde 3-phosphate decreased and there was no significant change in Vmax. Addition of Triton X-100 abolished the effect of both positively and negatively charged liposomes on the kinetic properties of the enzyme.     

Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase

Transient kinetic studies of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase show that all four sites of the tetrameric lobster enzyme are simultaneously active, apparently with equal reactivity. The rate-determining step of the oxidative phosphorylation is NADH release at high pH and phosphorolysis of the acyl-enzyme at low pH. For the reverse reaction the rate-determining step is a process associated with NADH binding, probably a conformation change, at high pH and d-glyceraldehyde 3-phosphate release at low pH. NADH has previously been shown to be a competitive inhibitor of the enzyme with respect to d-glyceraldehyde 3-phosphate and vice versa. This is consistent with the mechanism deduced from transient experiments given the additional proviso that 1-arseno-3-phosphoglycerate has a half-life of about 1min or longer at pH7. The dissociation constants of d-glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate to the NAD(+)-bound enzyme are too large to measure but are nevertheless consistent with the low K(m) values of these substrates.     

The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa.

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.     

Stereospecificity of C4 nicotinamide hydrogen transfer of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The stereospecificity of the reaction catalysed by the spinach chloroplast enzyme NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) with respect to the C4 nicotinamide hydrogen transfer was investigated. NADPH deuterated at the C4 HA position was synthesized using aldehyde dehydrogenase. 1H-NMR spectroscopy was used to examine the NADP+ product of the GPDH reaction for the presence or absence of the C4 deuterium atom. Chloroplast NADP-dependent glyceraldehyde-3-phosphate dehydrogenase retains the deuterium at the C4 HA position (removing the hydrogen atom), and is therefore a B (pro-S) specific dehydrogenase.     

Reaction of 5-enol-pyruvoylshikimate-3-phosphate synthase with diethyl pyrocarbonate: evidence for an essential histidine residue

5-enol-Pyruvoylshikimate-3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enol-pyruvoylshikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). Diethyl pyrocarbonate inactivated this enzyme with a second-order rate constant of 220 M-1 min-1 at pH 7.0 and 0 degrees C. The rate of inactivation is pH dependent and the pH inactivation rate data show the involvement of a group with a pKa of 6.8. Almost all of the original activity was recovered by treatment of the inactivated enzyme with hydroxylamine. The difference spectrum of the inactivated and native enzyme reveals a single peak at 242 nm but no trough at around 278 nm is observed. Complete inactivation required the modification of four histidine residues per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification shows that among the four modifiable residues, only one is critical for activity. Furthermore, this inactivation is prevented by the substrates of the enzyme. The above results indicated that one histidine is located within or very close to the active site and may play an important role in catalysis.     

Covalent binding of 3-pyridinealdehyde nicotinamide adenine dinucleotide and substrate to glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phoshate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12) forms a complex with 3-pyridinealdehyde-NAD which survives precipitation with 7% perchloric acid. The molar ratio bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD. 3-Pyridinealdehyde-deamino-NAD or glyceraldehyde 3-phosphate also forms an acid-stable complex with glyceraldehyde 3-phosphate dehydrogenase; however, NAD, 3-acetylpyridine-NAD, or thionicotinamide-NAD does not produce an acid-stable complex. Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by[14-C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphates activity of the enzyme for which the active site sulfhydryl residues must be oxidized.     

Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.     

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.     

Ion-pair formation as a source of enhanced reactivity of the essential thiol group of D-glyceraldehyde-3-phosphate dehydrogenase.

The reactivity and the mode of activation of the essential--SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins. Alkylations of Cys-149 of NAD-free D-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys-149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK1 equals 5.5, pK2 equals 8.2). It is concluded that there are two reactive forms of the--SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176. The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion. The two reactive forms are transformed into each other with pK2 equals 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK1 equals 5.5. In the presence of NAD, only the pH-rate profile of alkylation of D-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion-pair exists in a somewhat wider pH-range in the enzyme-coenzyme complex. An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.     

Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)- [NAD(P)-] dependent aldehyde dehydrogenases share a number of conserved amino acid residues, several of which are directly implicated in catalysis. In the present study, the role of Glu-268 from nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans was investigated. Its substitution by Ala resulted in a k(cat) decrease by 3 orders of magnitude. Pre-steady-state analysis showed that, for both the wild-type and E268A GAPNs, the rate-limiting step of the reaction is associated with deacylation. The pH dependence of the rate of acylation of wild-type GAPN is characterized by the contributions of distinct enzyme protonic species with two pK(a)s of 6.2 and 7.5. Substitution of Glu-268 by Ala resulted in a monosigmoidal pH dependence of the rate constant of acylation with a pK(a) of 6.2, which suggested the assignment of pK(a) 7.5 to Glu-268. Moreover, the E268A substitution did not significantly affect the efficiency of acylation of GAPN, showing that Glu-268 is not critically involved in the acylation, which includes Cys-302 nucleophilic activation and hydride transfer. On the contrary, the drastic decrease of the steady-state rate constant for the E268A GAPN demonstrated the essential role of Glu-268 in the deacylation. At basic pH, the solvent isotope effect of 2.3, characterized by a unique pK(a) of 7.7, and the linearity of the proton inventory showed that the rate-limiting process for deacylation is associated with the hydrolysis step and suggested that the glutamate form of Glu-268 acts as a base catalyst in this process. Surprisingly, the double-sigmoidal form of the pH-steady-state rate constant profile, characterized by pK(a) values of 6.1 and 7.4, revealed the high efficiency of the deacylation even at pH lower than 7.4. Therefore, we propose that the major role of Glu-268 is to promote deacylation through activation and orientation of the attacking water molecule, and in addition to act as a base catalyst at basic pH. From these results in relation to those recently described [Marchal, S., and Branlant, G. (1999) Biochemistry 38, 12950-12958], a scenario for the chemical catalysis of GAPN is proposed.     

Adsorption of glyceraldehyde 3-phosphate dehydrogenase on condensed monolayers of phospholipid.

The adsorption of [14C] alkylated glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle to condensed monolayers of phosphatidic acid was investigated under a variety of conditions. 2. The rate constant for association at 20 degrees C depended on ionic strength. At I/2=60mM the rate constant was 0.39min-1. At I/2=260mM it decreased to 0.27min-1. 3. The apparent association constant (Kass.) for adsorption at I/2=60mM was 1.06 X 10(6)M-1 and was strongly influenced by subphase changes in pH and ionic strength. Measurements of Kass. at 20 degrees and 5 degrees C gave a value for the apparent enthalpy change on adsorption of -33kJ-mol-1. Calculations of the apparent change in free energy and apparent entropy change for the adsorption process gave values of -34kJ-mol-1 and +2J-K-1-mol-1 respectively. 4. Decreasing the amount of phosphatidic acid in the monolayer by replacement with phosphatidylcholine caused the shape of the adsorption isotherm to change from apparent hyperbolic to sigmoid. Subphase changes in pH or ionic strength did not affect the shape of the adsorption isotherm. However, adsorption of enzyme on monolayers of 100% phosphatidic acid in the presence of 1mM-CaCl2 was sigmoid in nature. 5. It is concluded that glyceraldehyde 3-phosphate dehydrogenase binds to condensed charged monolayers by multiple electrostatic interactions. At low concentrations of phosphatidic acid in the monolayer or in the presence of Ca2+, this occurs in a two-step process and depends on lateral diffusion of phosphatidic acid for strong binding to take place.     

Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by fluorescein-5'-isothiocyanate: evidence that the dye participates in the interaction

An interaction of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled with FITC was studied by following the changes in fluorescence intensity of the bound dye. The association between the two enzymes was found to be a rather slow process characterized by a second order rate constant of 1.1 +/- 0.2.10(3) M-1 s-1, the KD of the complex between apoenzymes being 3.2.10(-7) M. The stability of the complex increased upon increase of temperature and ionic strength of the medium, suggesting a hydrophobic character of association. The ligands which bind at the active centers of the two enzymes (NAD+, ATP, 3-phosphoglycerate) weakened the bienzyme association. Unlabeled 3-phosphoglycerate kinase was unable to displace the FITC-labeled enzyme from the complex. Taken together, the results indicate that interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by FITC is assisted by the dye, which may bind at nucleotide-binding sites of GPDH. No interaction was observed between the FITC-labeled 3-phosphoglycerate kinase and lactate dehydrogenase, which suggests that protein-protein interaction at specific "recognition" sites may be a prerequisite for the complex formation.     

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.