The development of SS''-polymethylenebis(methanethiosulphonates) as reversible cross-linking reagents for thiol groups and their use to form stable catalytically active cross-linked dimers within glyceraldehyde 3-phosphate dehydrogenase.

The synthesis of a series of SS'-polymethylenebis(methanethiosulphonates) including the pentane, hexane, octane, decane and dodecane derivatives is described. These derivatives were synthesized by condensation between dibromoalkanes and potassium methanethiosulphonate in refluxing methanol and this seems an especially versatile reaction for the synthesis of asymmetric thiosulphonate derivatives. The synthesis of SS'-[1,8-3H4]-octamethylenebis(methanethiosulphonate) was also perfomed. Cross-linking was demonstrated in the four enzymes lactate dehydrogenase, phosphofructokinase, pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase. For all four enzymes cross-linking was efficiently reversed by reducing conditions in denaturing solvents. The reaction with glyceraldehyde 3-phosphate dehydrogenase was unique in that only the cross-linked dimer was produced in significant amounts (greater than 90% of total products as dimer). This reaction was followed in detail with radioactive cross-linking reagent. Inhibition of enzyme activity was extremely fast and showed an asymmetric distribution of enzyme activity on subunits. Thus complete modification of only one subunit resulted in up to 75% inhibition of enzyme activity. Reaction of glyceraldehyde 3-phosphate dehydrogenase with 1.25 mol of SS'-octamethylenebis(methanethiosulphonate) per mol of enzyme subunit produced two species of protein. The first species was obtained in 20% yield and was only partially re-activated on mild reduction with 2-mercaptoethanol. The second species was isolated in 66% yield and was completely re-activated on mild reduction. Before reduction there was 4 mol of inhibitor per tetramer for the latter species, and more than 95% of the enzyme was present as a dimer on non-reducing electrophoresis. After mild reduction 2 mol of inhibitor was still bound per tetramer, the enzyme was now catalytically active and the dimer was still the major structure on non-reducing electrophoresis. Thus mild reduction of SS'-octamethylenebis(methanethiosulphonate-treated glyceraldehyde 3-phosphate dehydrogenase enabled the production of active enzyme in which there is a stable cross-link across one of the molecular axes of the tetrameric enzyme. This cross-link was only reversed if reduction was performed when the enzyme was denatured. The molecular weight of cross-linked and re-activated cross-linked glyceraldehyde 3-phosphate dehydrogenase was established as 144000 (tetramer) by sucrose-density-gradient centrifugation. These observations are interpreted in terms of the molecular structure of glyceraldehyde 3-phosphate dehydrogenase.     

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.     

Covalent cross-linking of proteins without chemical reagents

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.     

Reactive lysines of yeast glyceraldehyde 3-phosphate dehydrogenase. Attachment of a reporter group to a specific non-essential residue

The lysine-183 residues of yeast glyceraldehyde 3-phosphate dehydrogenase, in contrast to the cysteine-149 residues, react independently with acylating and alkylating agents. Modification of all four residues is required to inactivate the enzyme in spite of the fact that this residue is apparently in the neighborhood of the cysteine-149 involved in half-of-the-sites activity. The modification of the lysine-183 residue, however, influences the half-of-the-sites effect since alkylation of the cysteine-149 residues of the enzyme whose lysine-183 residues are acetylated follows a linear pattern with each subunit acting independently. Four lysine residues outside the active site can be modified with fluorodinitrobenzene, causing 80% loss in enzyme activity. Once again each subunit acts independently. This same residue can also be modified by a fluorescein label which can serve as a reporter group for binding and conformational changes occurring at the active site. The results add support for the functional symmetry of the apo-enzyme and demonstrate how the co-operativity between subunits can be altered by amino acid modification.     

Formation of a polymethylene bis(disulfide) intersubunit cross-link between cysteine-281 residues in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase using octamethylene bis(methane[35S]thiosulfonate)

The synthesis of a radioactive cross-linking agent, S,S'-octamethylene bis(methane[35S]thiosulfonate) (OBMTS), is described. The route of synthesis can be generally used in the synthesis of 35S-labeled thiosulfonates for the selective modification of thiols in proteins. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) reacts asymmetrically with the bifunctional inhibitor. Initially two molecules of OBMTS react rapidly with the active-site thiol, Cys-149, on two of the four subunits to inhibit the enzyme completely without cross-linking. This is followed by the modification of four Cys-281 residues to incorporate two cross-links into the tetramer. Reduction of modified G3PD with 5 mM dithioerythritol under nondenaturing conditions released the inhibitor blocking the active-site thiol and completely restored enzyme activity while leaving the cross-link intact. Sodium dodecyl sulfate (Na-DodSO4) gel electrophoresis of the cross-linked enzyme under nonreducing conditions showed a dimer (Mr 72000) as the major species which was only cleaved by reduction in Na-DodSO4 containing beta-mercaptoethanol. The monomer formed was still radioactive, showing that the first disulfide in the cross-link was reduced at a much faster rate than the second disulfide. The latter was only reduced by using vigorous conditions. The location of the intersubunit cross-linked residues was established by isolation of the cyanogen bromide and tryptic subdigest peptides containing modified Cys-281. There were identified by molecular weight, amino terminal sequence, and amino acid composition.     

Molecular origin of the aging effects in glyceraldehyde-3-phosphate dehydrogenase

Rat muscle glyceraldehyde-3-phosphate dehydrogenase was reacted with two reagents aimed at the highly reactive cysteine-149 residue in the active site of the enzyme. The enzyme was rapidly inactivated by iodine monochloride. Complete inactivation occured when approx. 6 mol ICl were added per mol enzyme, indicating that reactions which compete with the reagent's interaction with cysteine-149 take place. Iodine was also found to inactivate the enzyme rapidly and effectively, and, when not in excess, this reagent interacted specifically with cysteine-149. The fraction of original enzymatic activity which could be restored by 2-mercaptoethanol in enzyme samples inhibited by 4.2 mol I₂/mol enzyme, decreased with time to a limiting value of 0.6 reached after approx. 15 min. The enzyme thus treated showed a remarkable similarity to enzyme samples purified from old rats, both in its activity and in NAD⁺ binding patterns under various conditions. It is concluded that the structural modifications induced in the modified enzyme resemble the age-related modifications in native ‘old’ enzyme. These results demonstrate that the origin of the age-related effects in glyceraldehyde-3-phosphate dehydrogenase is in subtle, post-synthetic structural changes. The inactivation reactions described above require a non-reducing environment for the enzyme. Whether such conditions do exist in cells of old animals is the subject of future studies.     

The comparative structure of mammalian glyceraldehyde 3-phosphate dehydrogenase

1. The amino acid sequences around the thiol groups of glyceraldehyde 3-phosphate dehydrogenase from badger and monkey skeletal muscle were compared with the sequences around the thiol groups in the enzyme isolated from other organisms. 2. Preliminary evidence of the existence of isoenzymes in the badger was obtained. Only the major form, however, could be purified completely. 3. The monkey enzyme contains only three cysteine residues per polypeptide chain compared with the four found in all the other mammalian enzymes so far examined, including that of badger, and the two in yeast. The missing thiol group in monkey was identified as residue 281 in the corresponding sequence of the pig enzyme. 4. These experiments rule out any essential role for cysteine-281 in the function of the mammalian enzymes. 5. Further evidence of the remarkable conservation of amino acid sequence in this enzyme during evolution is presented and discussed.     

Specific modification of a single cysteine residue in both bovine liver glutamate dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase. Difference in the mode of modification by pyrene maleimide

Pyrene maleimide is shown to be a 'half of the sites' reagent for glutamate dehydrogenase and for glyceraldehyde-3-phosphate dehydrogenase. The modified residues are identified as cysteine-115 for glutamate dehydrogenase and cysteine-149 for glyceraldehyde-3-phosphate dehydrogenase. The two enzymes react differently with pyrene maleimide. Whereas the hydrophobic environment of cysteine-115 directs the modification of glutamate dehydrogenase, the high reactivity of cysteine-149 determines the specific modification of glyceraldehyde-3-phosphate dehydrogenase. Glutamate dehydrogenase activity is unaltered by the modification: glyceraldehyde-3-phosphate dehydrogenase activity in inhibited.     

New protein cross-linking reagents that are cleaved by mild acid

New homo- and heterobifunctional cross-linking reagents have been synthesized. These reagents are based on ortho ester, acetal, and ketal functionalities that undergo acid-catalyzed dissociation but are base stable. The protein-reactive group in all the homobifunctional reagents is a maleimide group; the heterobifunctional acetal cross-linker has a maleimide group at one end and an N-hydroxysuccinimide ester at the other. These reagents have been used to cross-link diphtheria toxin (DT) to itself to give covalently cross-linked DT dimer or to conjugate DT monomer to the anti-CD5 antibody, T101. The hydrolysis of these cross-linked proteins was studied as a function of pH. Cleavage rates vary from minutes to hours at the pH of acidified cellular vesicles (approximately pH 5.4), ortho esters being the fastest, acetals the slowest, and ketals intermediate, but the cross-linked products are approximately 100 times more stable at the vascular pH of 7.4 and 1000 times more stable at a storage pH of 8.4 in all cases. The utility of these reagents in the reversible blockade of a toxic protein functional domain was demonstrated by using cross-linked DT dimer where the blocking and unblocking of toxin binding sites correlates with cellular toxicity. Of the different cross-linkers described, the acetone ketal, bis(maleimidoethoxy)propane (BMEP), appears to be the most promising in the construction of highly efficacious immunotoxins.     

Chemical cross-linking study of complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans

Two soluble periplasmic redox proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase and the copper protein amicyanin, form a weakly associated complex that is critical to their physiological function in electron transport [Gray, K. A., Davidson, V. L., & Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990]. The specific interactions between methylamine dehydrogenase and amicyanin have been studied by using the water-soluble cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Treatment of methylamine dehydrogenase alone with EDC caused no intermolecular cross-linking but did cause intramolecular cross-linking of this alpha 2 beta 2 oligomeric enzyme. The primary product that was formed contained one large and one small subunit. Methylamine dehydrogenase and amicyanin were covalently cross-linked in the presence of EDC to form at least two distinct species, which were identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). The formation of these cross-linked species was dependent on ionic strength, and the ionic strength dependence was much greater at pH 6.5 than at pH 7.5. The effects of pH and ionic strength were different for the different cross-linked products. SDS-PAGE and Western blot analysis of these cross-linked species indicated that the primary site of interaction for amicyanin was the large subunit of methylamine dehydrogenase and that this association could be stabilized by hydrophobic interactions. In light of these results a scheme is proposed for the interaction of amicyanin with methylamine dehydrogenase that is consistent with previous data on the physical, kinetic, and redox properties of this complex.     

Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.     

Application of zero-length cross-linking to form lysozyme, horseradish peroxidase and lysozyme-peroxidase dimers: Activity and stability

A facile method for the formation of covalent bonds between protein molecules is zero-length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, the cross-linking is performed for lysozyme, peroxidase (a glycoprotein) and between lysozyme–peroxidase by the method of Simons et al. [B.L. Simons, M.C. King, T. Cyr, M.A. Hefford, H. Kaplan, Covalent cross-linking of protein without chemical reagents, Protein Sci. 2002, 11, 1558–1564]. Approximately one-third of the total lysozyme becomes cross-linked and the dimer form was the major product for both enzymes. This modification induced some changes in the kinetic properties of the dimer peroxidase, as evident by two-fold increasing of V_max compared to the monomer but the enzymatic activity of cross-linked lysozyme dimer was the same as monomer. The activity of lysozyme dimer remained constant up to 10 min at 80 °C, while peroxidase activity of both monomer and dimer began to decrease after heating. The structural changes of the enzymes were investigated by circular dichroism and intrinsic fluorescence techniques. Near UV result showed lysozyme possess a compact structure in the dimer form but disruption of tertiary structure of peroxidase dimer was observed. Also conformational changes were detected and discussed by intrinsic fluorescence experiments. Effect of several metals in the formation of lysozyme dimer showed that Co²⁺ is the most effective one but its effect was marginal. At the end formation of heterogeneous dimer, peroxidase–lysozyme, was achieved using this method.     

Cross-linking of the enzymes in the glycosome of Trypanosoma brucei

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.     

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Heterobifunctional cross-linking reagents lead to photodependent and photoindependent cross-linking

Heterobifunctional cross-linking reagents have been introduced into the catalytic subunit of cAMP-dependent protein kinase as potential probes for identifying specific points of contact between the catalytic (C)-subunit and the type II regulatory (RII) subunit in the holoenzyme complex. Since at least one of the 2 cysteine residues in the C-subunit is known to be in close proximity to the interaction site between the C-subunit and the RII-subunit, these cysteines were chosen initially as targets for covalent modification by two heterobifunctional cross-linking reagents, p-azidophenacyl bromide and N-4-(azidophenylthio)phthalimide. Treatment of the C-subunit with each reagent led to the stoichiometric modification of Cys-199 and Cys-343. In each case, the modified C-subunit was still capable of forming a stable complex with the RII-subunit. Both modified C-subunits also could be covalently cross-linked to the RII-subunit; however, the mechanisms for cross-linking differed. Catalytic subunit modified by p-azidophenacyl bromide was cross-linked to the RII-subunit in a photodependent manner by a mechanism that was maximal when holoenzyme was formed and cAMP was absent. In contrast, the C-subunit modified by N-4-(azidophenylthio)phthalimide was cross-linked to the RII-subunit by a mechanism that was independent of photolysis. In this case, cross-linking was enhanced by the presence of cAMP. This cross-linking was the result of a disulfide interchange between a modified cysteine in the C-subunit and an unmodified cysteine in the RII-subunit.     

Catalytic mechanism and interactions of NAD+ with glyceraldehyde-3-phosphate dehydrogenase: correlation of EPR data and enzymatic studies

Perdeuterated spin label (DSL) analogs of NAD+, with the spin label attached at either the C8 or N6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and conformational changes of the DSL-NAD+-enzyme complex during the catalytic reaction. C8-DSL-NAD+ and N6-DSL-NAD+ showed 80 and 45% of the activity of the native NAD+, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism of this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD+ in three conditions: (1) DSL-NAD+ freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD+ tightly bound to enzyme subunits remote (58 A) from other NAD+ binding sites, and (3) DSL-NAD+ bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 A or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD+ in these three environments and measurements of the binding constants, K1-K4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD+ to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraldehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 A) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD+ complex. When glyceraldehyde-3-phosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the phosphoglyceryl residue and DSL-NAD+. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD+. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD+ from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.     

Dimeric enzyme IImtl of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. Cross-linking studies with bifunctional sulfhydryl reagents

The occurrence of intermolecular dithiols on EIImtl has been studied with a number of thiol-specific cross-linking reagents. The reaction of EIImtl with bifunctional maleimide derivatives inactivates the enzyme. At the same time the enzyme is irreversibly cross-linked to a dimeric species. Under optimal conditions 50% of the protein is cross-linked upon reaction with the dimaleimides. The enzyme is also cross-linked under oxidizing conditions in the presence of CuCl2, presumably by oxidizing an intermolecular dithiol to a disulfide. This oxidation can be reversed by the addition of the reducing agent dithiothreitol. The reaction of phosphorylated EIImtl with the same sulfhydryl-specific bifunctional reagents does not lead to any cross-linked product. The results are discussed in terms of the association state of the purified protein and the distribution of its thiol groups.     

Half-of-the sites reactivity and negative co-operativity: the case of yeast glyceraldehyde 3-phosphate dehydrogenase

Covalent modification of two of the four cysteine-149 residues of yeast glyceraldehyde 3-phosphate dehydrogenase, at pH 8.5, decreases the reactivity of the remaining two cysteine-149 residues and essentially inactivates the protein. The structure of the modifying reagent has only a secondary influence on this half-of-the-sites effect. Reactivity studies, together with the existing X-ray and sequence studies, suggest that the four active sites are initially functionally identical both in activity and in cysteine reactivity. The half-of-the-sites effect therefore arises in part or in whole from ligand-induced negatively co-operative conformational changes. A detailed kinetic study with iodoacetamide gives relative values of two rapidly reacting groups, a third more slowly reacting, and a fourth very slowly reacting group. These data, added to the existing data on cytidine triphosphate synthetase and alkaline phosphatase, suggest that the half-of-the-sites phenomena in many enzymes may be explained by ligand-induced negative co-operativity triggered by binding or covalent bond formation or both.     

Bovine lens aldehyde dehydrogenase. Kinetics and mechanism.

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.     

Glyceraldehyde 3-phosphate dehydrogenase of Scenedesmus obliquus: Promotion of NADPH-dependent activity and antagonisation by NAD

The glyceraldehyde 3-phosphate dehydrogenase activity of extracts from heterotrophic Scenedesmus obliquus was linked predominantly to NADH. However, on DEAE-cellulose chromatography the enzyme was eluted by a gradient of phosphate in a form characterized by high NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity. This interconversion of enzyme forms could be prevented by the presence of NAD during DEAE-cellulose chromatography.High concentrations of phosphate stimulated the NADPH-dependent activity of the purified enzyme at the expense of activity linked to NADH and these changes were associated with depolymerization of a hexadecamer to a tetramer. The effect of phosphate on the rates of increase in NADPH-dependent activity and of a decrease in activity linked to NADH was cooperative with a Hill coefficient of 3.2. The inversely related changes in coenzyme specificity were inhibited to the same extent by NAD and the response to this ligand was anticooperative. These findings imply a strictly inverse proportional relationship between the rates of change of NADH and NADPH-linked activity. In the presence of dithiothreitol, low concentrations of phosphate promoted NADPH-dependent activity by stabilising the unstable tetrameric form produced from the hexadecamer by the thiol.These phenomena are discussed in relation to a general mechanism for the in vivo promotion of NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity.     

A new chemical mechanism catalyzed by a mutated aldehyde dehydrogenase.

NAD(P) aldehyde dehydrogenases (EC 1.2.1.3) are a family of enzymes that oxidize a wide variety of aldehydes into acid or activated acid compounds. Using site-directed mutagenesis, the essential nucleophilic Cys 149 in the NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli has been replaced by alanine. Not unexpectedly, the resulting mutant no longer shows any oxidoreduction phosphorylating activity. The same mutation, however, endows the enzyme with a novel oxidoreduction nonphosphorylating activity, converting glyceraldehyde 3-phosphate into 3-phosphoglycerate. Our study further provides evidence for an alternative mechanism in which the true substrate is the gem-diol entity instead of the aldehyde form. This implies that no acylenzyme intermediate is formed during the catalytic event. Therefore, the mutant C149A is a new enzyme which catalyzes a distinct reaction with a chemical mechanism different from that of its parent phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This finding demonstrates the possibility of an alternative route for the chemical reaction catalyzed by classical nonphosphorylating aldehyde dehydrogenases.