Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.     

The functional identity of the active centres of transketolase.

Direct determination of the number of catalytically active molecules of the coenzyme in holotransketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycoaldehydetransferase, EC 2.2.1.1) has corroborated our previous data indicating that in the native enzyme there are two active centres. They have been provided to be functionally identical. It has been shown that the decrease in the specific activity of transketolase during its storage is due to inactivation of one of the active centres, having a lower affinity for the coenzyme. The second active centre retains thereby its full catalytic activity.     

Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae.

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.     

The sterochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate 6-phosphate and 2-keto-3-deoxygalactonate-6-phosphate aldolase of Pseudomonas saccharophila.

In Pseudomonas saccharophila 2-keto-3-deoxygalactonate-6-P aldolase (EC 4.1.2.21) is induced by growth on galatose while 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) is constitutive. These enzymes catalyze identical reactions except for the configuration fixed at C-4 during the condensation reaction. It was found with each enzyme that in a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P, the respective condensation products were formed 8 to 10 times faster than tritium was released to water. Since pyruvate deprotonation is obligatory for condensation, the above result requires a hydrogen isotope effect in enolpyruvate formation, which must be then at least partially rate limiting for C--C synthesis. Further, condensation between D-glyceraldehyde-3-P and (3R)-[3-3H, 2H,H]pyruvate or (3S)-[3-3H, 2H,H]pyruvate, as catalyzed by each enzyme, enriched for (3R)- and (3S)-3-3H, 2H-labeled condensation product, respectively. Thus, each enzyme catalyzes C--C and C--H synthesis with retention of configuration at C-3. This shows that the active sites of both enzymes are asymmetric since solutes can only approach a single face of the bound pyruvyl enolate. In addition, the respective aldehyde specific portions of the two active sites must have opposite chiralities, with respect to each other, for correctly orienting the carbonyl faces of the incoming D-glyceraldehyde-3-P, to generate the correct configuration at C-4 of the respective condensation products.     

The affinity chromatography of transketolase.

A number of possible affinity adsorbents for transketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphateglycolaldehydetransferase, EC 2.2.1.1) were prepared. The behaviour of the enzyme from Candida utilis and from Baker's yeast on columns of these and of Blue Sepharose CL-6B was examined, together with the behaviour of the contaminating enzyme, ribulose 5-phosphate 3-epimerase (EC 5.1.3.1). A procedure for removing bound thiamine pyrophosphate by dialysis against EDTA was developed. The competitive inhibition of transketolase by oxythiamine and neopyrithiamine was measured and the Ki values obtained of 1.4 and 4.3 mM, respectively, were compared with the affinity of adsorbents prepared from these two inhibitors. Adsorbents containing bound thiamine pyrophosphate were relatively ineffective but those containing epoxy-linked neopyrithiamine and D-ribose 5-phosphate adsorbed the enzyme at pH 7.4 and it could be eluted in a specific manner.     

Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.     

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.     

Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase and its functional consequences.

E. coli D-glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E. coli 3-phosphoglycerate kinase with a stoichiometry of 1.77 +/- 0.61 kinase molecules per tetramer of the dehydrogenase and an apparent Kd of 1.03 +/- 0.68 microM (10 mM sodium phosphate, 0.15 M NaCl). No interaction was detected between E. coli D-glyceraldehyde-3-phosphate dehydrogenase and rabbit muscle 3-phosphoglycerate kinase. The species-specificity of the bienzyme association made it possible to develop a kinetic approach to demonstrate the functionally significant interaction between E. coli D-glyceraldehyde-3-phosphate dehydrogenase and E. coli 3-phosphoglycerate kinase, which consists of an increase in steady-state rate of the coupled reaction.     

Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

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).     

Comparison of inactivation and conformational changes of D-glyceraldehyde-3-phosphate dehydrogenase during thermal denaturation

The inactivation of D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) (GAPDH) during thermal denaturation has been compared to its dissociation-aggregation measured by light scattering and changes in secondary structure measured by CD in the far ultraviolet. The inactivation at 38.5 degrees C consists of two stages. The rate of the first stage is too fast to be followed by conventional methods. The extent of this fast stage inactivation increases with increasing temperature and, more markedly, with increasing pH. At this stage, the inactivation is reversible and no appreciable dissociation or change in secondary structure can be detected. The secondary structure of the enzyme is relatively heat stable, showing no appreciable change at 38.5 degrees C. At this temperature, the enzyme first dissociates within several minutes probably into dimers and with prolonged heating, it becomes irreversibly aggregated. The above results are in accord with the earlier suggestion, based on results obtained during denaturation of a number of enzymes by guanidine hydrochloride (GdnHCl) and urea, that for some enzymes the active site is situated in a region more susceptible to perturbation than the molecule as a whole (Tsou, C.-L. (1986) Trends Biochem. Sci. 11, 427).     

The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.     

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.     

Glyceraldehyde-3-phosphate dehydrogenase of rat erythrocytes has no membrane component

In the course of studying mammalian erythrocytes we noted prominent differences in the red cells of the rat. Analysis of ghosts by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed that membranes of rat red cells were devoid of band 6 or the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). Direct measurements of this enzyme showed that glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was about 25% of that in human cells; all of the glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was within the cytoplasm and none was membrane bound; and in the human red cell, about 1/3 of the enzyme activity was within the cytoplasm and 2/3 membrane bound. The release of glyceraldehyde-3-phosphate dehydrogenase from fresh rat erythrocytes immediately following saponin lysis was also determined using the rapid filtration technique recently described. The extrapolated zero-time intercepts of these reactions confirmed that, in the rat erythrocyte, none of the cellular glyceraldehyde-3-phosphate dehydrogenase was membrane bound. Failure of rat glyceraldehyde-3-phosphate dehydrogenase to bind to the membranes of the intact rat erythrocyte seems to be due to cytoplasmic metabolites which interact with the enzyme and render it incapable of binding to the membrane.     

Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus

The D-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the D-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD+ and NADP+ and is not inhibited by pentalenolactone. The enzyme is intrinsically stable up to 75 degrees C. It is stabilized by the coenzyme NADP+ and at high ionic strength up to about 90 degrees C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52 degrees C.     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxidation of C1-compounds in Pseudomonas C.

Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.