SH-containing compounds as allosteric effectors of glyceraldehyde-3-phosphate dehydrogenase.

The rate of hydrolysis of 3-phosphoglyceroyl-holoenzyme, a covalent intermediate of glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction, is considerably decreased in the presence of micromolar concentrations of reduced glutathione, cysteine or dithiothreitol with Ki values of 0.78 microM, 0.6 microM and 10 microM, respectively. The maximal effect is achieved at a molar ratio [effector]/[tetrameric enzyme] close to unity, which points to subunit cooperatively involved in the stabilization of the covalent intermediate against hydrolysis. The effect is specific for acylholoenzyme conformation and insignificant in the case of hydrolysis of acylated apoenzyme species. The ability of the effectors to stabilize the reaction intermediate against spontaneous hydrolysis, in which water replaces inorganic phosphate as the acyl group acceptor, may be a factor contributing to the specificity and effectiveness of the enzyme catalysis.

D-glyceraldehyde-3-phosphate dehydrogenase: pre-existent asymmetry of the tetramer and its functional implications.

Modification of a single arginine residue per subunit of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase stabilizes the tetramer in a conformation wherein only two active sites are capable of performing catalysis (oxidation of D-glyceraldehyde 3-phosphate or hydrolysis of p-nitrophenyl acetate). The modified enzyme exhibits half-of-the sites reactivity towards iodoacetate and iodoacetamide, known to be 'all-of-the-sites reagents' with the native enzyme. Evidence is presented supporting the model of a built-in asymmetry of the tetramer. The results obtained suggest that the arginine residue (probably Arg-231) controls the conformational transition between the asymmetric and symmetric states of the tetramer.

Rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase: half-of-the-sites reactivity of the enzyme modified at arginine residues.

Modification of a single arginine residue per subunit of rabbit muscle apo-D-Glyceraldehyde-3-phosphate dehydrogenase does not affect the rate of hydrolysis of p-nitrophenyl acetate catalyzed by the enzyme, but locks the tetramer in a conformation wherein only two active sites are functioning. The modified enzyme also exhibits half-of-the sites reactivity towards iodoacetate and iodoacetamide. On the other hand, its NAD(+)-binding characteristics remain unchanged. Evidence is presented supporting the view that mechanisms of half-of-the-sites reactivity and negative cooperativity in coenzyme binding are different. The results are consistent with a built-in asymmetry of the tetramer and suggest that the arginine residue (probably Arg-231) controls the conformational transition between the asymmetric and symmetric states of the tetramer.

Rabbit muscle tetrameric D-glyceraldehyde-3-phosphate dehydrogenase is locked in the asymmetric state by chemical modification of a single arginine per subunit.

Modification of a single arginine residue per subunit of rabbit muscle tetrameric D-glyceraldehyde-3-phosphate dehydrogenase by 2,3-butanedione converts the enzyme into the form which retains 5-7% of the original activity and manifests cooperative properties that are absent in the native enzyme. It exhibits half-of-the-sites reactivity towards the natural substrate D-glyceraldehyde-3-phosphate. Titration of the modified enzyme with DTNB reveals only two instantaneously reacting SH groups, the total amount of SH groups approaching nine per tetramer. In the presence of 8 M urea, an additional seven SH groups become accessible to DTNB. This suggests that the arginine modification imposes some conformational constraints which affect the microenvironment of the active site cysteine residues in two subunits of the tetramer. The changes do not influence the interaction between the essential cysteine residue and NAD+ which is responsible for the change transfer complex formation, since the molar extinction coefficient of the apoenzyme-NAD+ complex, epsilon 360, was not altered upon the arginine modification. The native and close to four in the case of the native enzyme and about three with the modified one. The apparent pK values of Cys-149 within the functioning active centers of the tetramer were determined from the pH profiles of the inactivation rates in presence of iodoacetamide. The apparent pKa of the essential thiols was found to change upon enzyme modification from 9.44 to 10.07 in the apoenzyme and from 9.17 to 9.36 in the holoenzyme. The apparent pKa of the arginine residue determined from the pH dependence of the inactivation rate was equal to 9.0 and did not change upon apo-holo enzyme transition.

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).