Two glyceraldehyde-3-phosphate dehydrogenase isozymes from the koningic acid (heptelidic acid) producer Trichoderma koningii

The sesquiterpene lactone koningic acid (heptelidic acid) irreversibly inactivated glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde 3-phosphate: NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) (GAPDH) and thus inhibits glycolysis. The koningic-acid-producing strain of Trichoderma koningii M3947 was shown to contain the koningic-acid-resistant GAPDH isozyme (GAPDH I) under conditions of koningic acid production. In peptone-rich medium, however, no koningic acid production was observed, and the koningic-acid-sensitive GAPDH isozyme (GAPDH II), in addition to the resistant enzyme, was produced. Both enzymes were tetramer with a molecular mass of 152 kDa (4 x 38 kDa) and lost enzyme activity when two of the four cysteine residues reacted with koningic acid. The apparent Km values of GAPDH I and II for glyceraldehyde 3-phosphate were 0.54 mM and 0.33 mM, respectively. The former isozyme was inhibited 50% by 1 mM koningic acid but not affected at 0.1 mM, while the latter isozyme was inhibited 50% at 0.01 mM. The immunochemical properties and partial amino acid sequences suggested that the two isozymes have different molecular structures. These results suggest that GAPDH I is responsible for the glycolysis in T. koningii when koningic acid is produced.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

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)     

The 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of Neurospora crassa is Zn2+-dependent.

We have demonstrated the co-purification in constant ratio of all five activities of the pentafunctional arom enzyme complex from Neurospora crassa. Progressive inactivation of the 3-dehydroquinate synthase component of the purified enzyme complex by chelating agents was blocked by the substrate, 3-deoxy-D-arabino-heptulosonate 7-phosphate, but not by the cofactor NAD+. Full activity was restored at Zn2+ concentrations as low as 0.05 nM. Atomic absorption data indicated that the intact enzyme complex contained 1 atom per subunit of tightly bound zinc. The arom 3-dehydroquinate synthase had a calculated turnover number of 19s-1, this being within the narrow range of values obtained for the other four activities of the intact multifunctional enzyme. The Km for 3-deoxy-D-arabino-heptulosonate 7-phosphate was 1.4 microM in a phosphate-free buffer; inorganic phosphate was a competitive inhibitor with respect to 3-deoxy-D-arabino-heptulosonate 7-phosphate.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone: kinetic and mechanistic studies

Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (1) resulted in time-dependent, irreversible inhibition of GAPDH. The kinetics of inactivation were biphasic, exhibiting an initial rapid phase and a slower second phase. Pentalenolactone methyl ester (2) also irreversibly inactivated GADPH, albeit at a slower rate and with a higher KI. The substrate glyceraldehyde-3-phosphate (G-3-P) afforded protection against inactivation by 1, whereas the presence of NAD+ in the incubation mixture stimulated the inactivation by increasing the apparent affinity of the enzyme for the inhibitor. In steady-state kinetic experiments, 1 acted as a competitive inhibitor of GAPDH with respect to G-3-P but exhibited uncompetitive inhibition with respect to NAD+. Inactivation of NAD+-free apo-GAPDH by 1 showed simple pseudo-first-order kinetics. By titrating the free thiol residues of partially inactivated GAPDH, it was found that both pentalenolactone and pentalenolactone methyl ester react with all four Cys-SH residues of the tetrameric GAPDH.     

Suicide inactivation of fructose-1,6-bisphosphate aldolase

2-Keto-4,4,4-trifluorobutyl phosphate (HTFP) was prepared from 3,3,3-trifluoropropionic acid. HTFP acts as an irreversible inhibitor of rabbit muscle aldolase: the loss of activity was time dependent and the inactivation followed a pseudo-first-order process. Values of 1.4 mM for the dissociation constant and 2.3 X 10(-2) s-1 for the reaction rate constant were determined. The kinetic constants do not depend on the enzyme concentration. No effect of thiols on the inactivation rate was detected. Only 1-2 mol of fluoride ions was liberated per inactivated subunit, indicative of a low partition ratio. Dihydroxyacetone phosphate protected the enzyme against the inactivation in a competitive manner, and glyceraldehyde 3-phosphate protected as if it formed a condensation product with HTPF. 5,5'-Dithiobis(2-nitrobenzoic acid) thiol titration showed the loss of one very reactive thiol group per enzyme subunit after inactivation. All those observations seem to agree with a suicide substrate inactivation of aldolase by HTPF.     

The role of arginine residues in the function of D-glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of apo-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle in the presence of butanedione is the result of modification of one arginyl residue per subunit of the tetrameric enzyme molecule. The loss of activity follows pseudo-first-order kinetics. NAD+ increases the apparent first-order rate constant of inactivation. The effect of NAD+ on the enzyme inactivation is cooperative (Hill coefficient = 2.3--3.2). Glyceraldehyde 3-phosphate protected the holoenzyme against inactivation, decreasing the rate constant of the reaction. At saturating concentrations of substrate the protection was complete. The Hill plot demonstrates that the effect is cooperative. This suggests that subunit interactions in the tetrameric holoenzyme molecule may affect the reactivity of the essential arginyl residues. In contrast, glyceraldehyde 3-phosphate had no effect on the rate of inactivation of the apoenzyme in the presence of butanedione. 100 mM inorganic phosphate protected both the apoenzyme and holoenzyme against inactivation. The involvement of the microenvironment of the arginyl residues in the functionally important conformational changes of the enzyme is discussed.     

Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.     

Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.     

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.     

The sulfhydryl content of L-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.     

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.     

The role of nicotinamide adenine dinucleotide in the inhibition of bovine liver S-adenosylhomocysteine hydrolase by neplanocin A

Neoplanocin A, a cyclopentenyl analog of adenosine, has been shown recently to be a tight binding inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), exhibiting a stoichiometry of one molecule of inhibitor per molecule of the enzyme tetramer (Borchardt, R. T., Keller, B. T., and Patel-Thombre, U. (1984) J. Biol. Chem. 259, 4353-4358). In the present study a detailed analysis was performed of the possible role of the enzyme-bound NAD+ in the inactivation of AdoHcy hydrolase by neplanocin A. The NAD+/NADH content was quantitated using a fluorescence technique. The native enzyme showed intrinsic fluorescence with an emission maximum at 460 nm when excited at 340 nm, partially due to NADH bound to the enzyme. It was found that the content of NAD+ and NADH in freshly prepared, native enzyme is equal, having a stoichiometry of two nucleotides per enzyme molecule (tetramer). In addition, it was observed that the enzymatic activity of the native enzyme can be increased by about 30% following preincubation with NAD+. Furthermore, it was demonstrated that the mechanism of inhibition of AdoHcy hydrolase by neplanocin A involves the reduction of enzymatically bound NAD+ to NADH. Catalytic activity of the inactivated enzyme could be fully recovered in a time-dependent manner by further incubation with NAD+ (but not NADH). It was also found that inhibition by neplanocin A does not involve dissociation of the bound NAD+ or NADH from the enzyme, but simply reduction of the NAD+ to NADH.     

Partial purification and some kinetic properties of glucose-6-phosphate dehydrogenase from Phycomyces blakesleeanus

Glucose-6-phosphate dehydrogenase from sporangiophores of Phycomyces blakesleeanus NRRL 1555 (-) was partially purified. The enzyme showed a molecular weight of 85 700 as determined by gel-filtration. NADP+ protected the enzyme from inactivation. Magnesium ions did not affect the enzyme activity. Glucose-6-phosphate dehydrogenase was specific for NADP+ as coenzyme. The reaction rates were hyperbolic functions of substrate and coenzyme concentrations. The Km values for NADP+ and glucose 6-phosphate were 39.8 and 154.4 microM, respectively. The kinetic patterns, with respect to coenzyme and substrate, indicated a sequential mechanism. NADPH was a competitive inhibitor with respect to NADP+ (Ki = 45.5 microM) and a non-competitive inhibitor with respect to glucose 6-phosphate. ATP inhibited the activity of glucose-6-phosphate dehydrogenase. The inhibition was of the linear-mixed type with respect to NADP+, the dissociation constant of the enzyme-ATP complex being 2.6 mM, and the enzyme-NADP+-ATP dissociation constant 12.8 mM.     

Affinity labeling of cofactor-binding region of human placental estradiol 17 beta-dehydrogenase by periodate-oxidized NADP+ (o-NADP+)

Periodate-oxidized NADP+ (o-NADP+), an analogue of the cofactors, is a reversible inhibitor of estradiol 17 beta-dehydrogenase in human placenta. Mode of the inhibition by o-NADP+ appeared to be competitive type (Ki = 0.84 microM) against NAD+ and non-competitive type (Ki = 1.13 microM) against estradiol, respectively. Treatment of the estradiol 17 beta-dehydrogenase with o-NADP+ resulted in time-dependent loss of the enzyme activity. The inactivation exhibited pseudo-first order kinetics (t1/2 = 15 min) and was protected by NAD+ and NADP+. On the other hand, periodate-oxidized ATP inactivated slightly the estradiol 17 beta-dehydrogenase. These results indicate that the residue(s) of lysines is located near the cofactor-binding region of estradiol 17 beta-dehydrogenase of human placenta.     

The interactions of 9,10-phenanthrenequinone with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a potential site for toxic actions

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate, one of the precursors for glycolytic ATP biosynthesis. The enzyme contains an active site cysteine thiolate, which is critical for its catalytic function. As part of a continuing study of the interactions of quinones with biological systems, we have examined the susceptibility of GAPDH to inactivation by 9,10-phenanthrenequinone (9,10-PQ). In a previous study of quinone toxicity, this quinone, whose actions have been exclusively attributed to reactive oxygen species (ROS) generation, caused a reduction in the glycolytic activity of GAPDH under aerobic and anaerobic conditions, indicating indirect and possible direct actions on this enzyme. In this study, the effects of 9,10-PQ on GAPDH were examined in detail under aerobic and anaerobic conditions so that the role of oxygen could be distinguished from the direct effects of the quinone. The results indicate that, in the presence of the reducing agent DTT, GAPDH inhibition by 9,10-PQ under aerobic conditions was mostly indirect and comparable to the direct actions of exogenously-added H2O2 on this enzyme. GAPDH was also inhibited by 9,10-PQ anaerobically, but in a somewhat more complex manner. This quinone, which is not considered an electrophile, inhibited GAPDH in a time-dependent manner, consistent with irreversible modification and comparable to the electrophilic actions of 1,4-benzoquinone (1,4-BQ). Analysis of the anaerobic inactivation kinetics for the two quinones revealed comparable inactivation rate constants (k(inac)), but a much lower inhibitor binding constant (K(i)) for 1,4-BQ. Protection and thiol titration studies suggest that these quinones bind to the NAD+ binding site and modify the catalytic thiol from this site. Thus, 9,10-PQ inhibits GAPDH by two distinct mechanisms: through ROS generation that results in the oxidization of GAPDH thiols, and by an oxygen-independent mechanism that results in the modification of GAPDH catalytic thiols.     

Evidence for a substrate assisted conformational transformation of glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase exhibits half-site reactivity, the structural origin of which is obscure. Thermal inactivation kinetics, employed here as a probe for site-site heterogeneity in solution, show that green gram glyceraldehyde 3-phosphate dehydrogenase (in the absence and presence of phosphate and NAD+) loses activity in two distinct phases, each of which accounts for half of the initial activity. In the presence of substrate, glyceraldehyde 3-phosphate the relative amplitude of the slow phase increases, and at 0.06 mM glyceraldehyde 3-phosphate the time-course of inactivation corresponds to a single exponential decay. The data are consistent with a suggestion that glyceraldehyde 3-phosphate dehydrogenase may exist in two interconvertible conformations of different symmetry characteristics (C2 in equilibrium D2). The lower symmetry conformation (C2) predominates in the apoenzyme and in the presence of phosphate and NAD+. The higher symmetry conformation (D2) is stabilised by glyceraldehyde 3-phosphate.     

4-Chloroacetylpyridine adenine dinucleotide. A highly reactive and chromophoric affinity label of glyceraldehyde-3-phosphate dehydrogenase from sturgeon

The analogue of NAD+, 4-chloroacetylpyridine-adenine dinucleotide (clac4PdAD+), inactivated the glyceraldehyde-3-phosphate dehydrogenase from sturgeon at a high rate. An affinity labeling was shown to occur with clac4PdAD+. The mononucleotide 4-chloroacetylpyridine 1-beta-D-ribose 5'-phosphate (clac4PdMN+) reacted with the enzyme in a second-order reaction whose rate was much smaller than that calculated for clac4PdAD+ taken as a second-order rate reagent. The rate of the reaction of clac4PdAD+ with the enzyme was determined by stopped flow, using as a probe the long-wavelength absorption maximum (430 nm) formed concomitantly with inactivation of the enzyme. Computer-assisted graphic simulation showed that the clac4PdAD+ analogue could bind to the active site of the enzyme from Bacillus stearothermophilus in a similar manner to that of NAD+, and that the reactive carbon and the reactive thiolate of Cys-149 were within bonding distance. The absorption at 430 nm was linearly proportional to the substoichiometric concentration of clac4PdAD+/mole subunit. Thiol titration suggested the modification of one thiol residue per subunit. The modified thiol was identified by degradation as Cys-149. In contrast to the absorption band generated during the reaction of the 3-chloroacetylpyridine-adenine dinucleotide (clac3PdAD+) with the same enzyme [Eur. J. Biochem. (1982) 127, 519-524; 129, 437-446], enzyme inactivated with clac4PdAD+ and clac4PdMN+ exhibited an absorption maximum at long wavelength which was still present after denaturation. The chromophore is proposed to be the enol form of the alpha-thioether ketone produced by alkylation of the thiolate of Cys-149 by the chloroacetyl group.     

Identification of koningic acid (heptelidic acid)-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The sesquiterpene antibiotic koningic acid (heptelidic acid) has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in specific manner, probably by binding to the sulfhydryl residue at the active site of the enzyme (Sakai, K., Hasumi, K. and Endo, A. (1988) Biochim. Biophys. Acta 952, 297-303). Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase labeled with [3H]koningic acid was digested with trypsin. Reverse-phase HPLC revealed that the label is associated exclusively with a tryptic peptide having 17 amino acid residues. Microsequencing and fast atom bombardment mass spectrometry demonstrated that the peptide has the sequence Ile-Var-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn-Cys-Leu-Ala-Pro-Leu-Ala-Lys. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue corresponding to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity, was shown to be the site modified by koningic acid. Structural analyses of the reaction product of koningic acid and L-cysteine suggested that the epoxide of koningic acid reacts with the sulfhydryl group of cysteine residue, resulting in a thioether.