The activation and inactivation of the acyl phosphatase activity of glyceraldehyde-3-phosphate dehydrogenase

doi:10.1016/0003-9861(70)90209-2

Archives of Biochemistry and Biophysics

Volume 136, Issue 2, February 1970, Pages 383-391

https://doi.org/10.1016/0003-9861(70)90209-2 Get rights and content

Abstract

When the dehydrogenase activity of muscle glyceraldehyde-3-phosphate dehydrogenase is reversibly inactivated with o-iodosobenzoate or under certain conditions with iodine monochloride, the enzyme is converted to an acetyl phosphatase. Inactivation of the dehydrogenase activity with tetrathionate, iodoacetate, and iodoacetamide does not convert the enzyme to an acetyl phosphatase. When the dehydrogenase activity is reversibly inactivated with o-iodosobenzoate at 0 ° and then incubated at 37 °, the dehydrogenase activity is irreversibly inactivated and the acetyl phosphatase activity disappears at a comparable rate. When CN⁻, SO₃⁼, S₂O₃⁼, thiourea, 6-propyl thiouracil, and phenyl arsene oxide are added to the enzyme at pH 5.9 after its oxidation with o-iodosobenzoate, the acetyl phosphatase activity is inactivated. Evidence which has been presented previously (14) suggests that the catalytically active sulfhydryl group of glyceraldehyde-3-phosphate dehydrogenase is converted to a stabilized sulfenic acid residue during its reversible inactivation with o-iodosobenzoate. To reconcile the activation and inactivation of the acetyl phosphatase activity by the various reagents used in this study, it is suggested that the stabilized sulfenic acid derivative of the catalytically active sulfhydryl group of glyceraldehyde-3-phosphate dehydrogenase participates directly in acetyl phosphatase hydrolysis. A mechanism which postulates a sulfenyl carboxylate intermediate has been proposed for the hydrolytic reaction.

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Citation Excerpt :
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Reactive oxygen species-mediated cysteine sulfenic acid modification has emerged as an important regulatory mechanism in cell signaling. The stability of sulfenic acid in proteins is dictated by the local microenvironment and ability of antioxidants to reduce this modification. Several techniques for detecting this cysteine modification have been developed, including direct and in situ methods.
This review presents a historical discussion of sulfenic acid chemistry and highlights key examples of this modification in proteins. A comprehensive survey of available detection techniques with advantages and limitations is discussed. Finally, issues pertaining to rates of sulfenic acid formation, reduction, and chemical trapping methods are also covered.
Early chemical models of sulfenic acid yielded important insights into the unique reactivity of this species. Subsequent pioneering studies led to the characterization of sulfenic acid formation in proteins. In parallel, the discovery of oxidant-mediated cell signaling pathways and pathological oxidative stress has led to significant interest in methods to detect these modifications. Advanced methods allow for direct chemical trapping of protein sulfenic acids directly in cells and tissues. At the same time, many sulfenic acids are short-lived and the reactivity of current probes must be improved to sample these species, while at the same time, preserving their chemical selectivity. Inhibitors with binding scaffolds can be rationally designed to target sulfenic acid modifications in specific proteins.
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Nitroxyl (HNO), the one-electron reduced and protonated congener of nitric oxide (NO), is a chemically unique species with potentially important biological activity. Although HNO-based pharmaceuticals are currently being considered for the treatment of chronic heart failure or stroke/transplant-derived ischemia, the chemical events leading to therapeutic responses are not established. The interaction of HNO with oxidants results in the well-documented conversion to NO, but HNO is expected to be readily reduced as well. Recent thermodynamic calculations predict that reduction of HNO is biologically accessible. Herein, kinetic analysis suggests that the reactions of HNO with several mechanistically distinct reductants are also biologically feasible. Product analysis verified that the reductants had in fact been oxidized and that in several instances HNO had been converted to hydroxylamine. Moreover, a theoretical analysis suggests that in the reaction of HNO with thiol reductants, the pathway producing sulfinamide is significantly more favorable than that leading to disulfide. Additionally, simultaneous production of HNO and NO yielded a biphasic oxidative capacity.
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The microsomal FAD-containing monooxygenase (EC 1.14.13.8, dimethylaniline monooxygenase) purified to homogeneity from hog liver catalyzes NADPH- and oxygen-dependent S-oxygenation of phenylthiourea, ethylenethiourea, thiocarbanilide, N-methylthiourea, and thiourea to their corresponding formamidine sulfinic acids. The sulfinic acids are formed by sequential enzymic oxidation of the thioureas through intermediate sulfenic acids. The reaction sequence was established by separating intermediate and final oxygenated metabolites of phenylthiourea and ethylenethiourea. The sulfenic and sulfinic acids of these two thioureas, produced enzymically, were chromatographically and spectrally identical with chemically synthesized reference compounds. Phenylformamidine and ethyleneformamidine sulfinic acids are slowly converted to their sulfonic acids upon prolonged incubation. While N-substituted formamidine sulfinic acids oxidize spontaneously to formamidine sulfonic acids at 37 °C, the further oxidation of ethyleneformamidine sulfinic acid may be, at least in part, enzyme catalyzed. The purified monooxygenase also catalyzes rapid oxygenation of mercaptoimidazoles to the corresponding imidazole sulfinic acids. The instability of S-oxygenated mercaptoimidazoles prevented their isolation and positive identification, but analysis of kinetic data obtained with sulfenic acid trapping agents suggests that these compounds are oxygenated by the same reaction sequence established for N-substituted thioureas. The NADPH- and oxygen-dependent oxidation of thiocarbamates and of 2-mercaptoimidazoles catalyzed by hog or hamster liver microsomes correlates with dimethylaniline N-oxidase activity and appears completely independent from cytochrome P-450. The S-oxidation of thiourea and its derivatives is not inhibited by n-octylamine, a known inhibitor of cytochrome P-450 dependent oxygenations. Furthermore, differential thermal inactivation of the flavin-containing monooxygenase totally abolishes phenylthiourea S-oxidase activity of hamster liver microsomes.
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Treatment of highly purified ovine erythrocyte glutathione peroxidase with KCN resulted in loss of enzyme activity and release of selenium from the enzyme. The inactivation by cyanide was time-dependent and the rate was strongly influenced by temperature, concentration of cyanide, oxidation state of the enzyme, and pH. The pH effect could be explained on the basis of the increasing proportion of cyanide ion with increasing pH. Inhibition could be prevented by prior reduction of the purified enzyme with glutathione, dithiothreitol, or dithionite. Oxidation with cumene hydroperoxide was necessary to demonstrate cyanide inhibition of glutathione peroxidase activity in rat liver cytosol. These observations explain why cyanide inhibition of glutathione peroxidase has not been noted previously and provide new approaches for studying the chemical nature of the enzyme-selenium.

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^☆: Publication No. 673 from the Graduate Department of Biochemistry, Brandeis University. Supported by Research Grant GM-14,481 from the National Institute of General Medical Sciences, N.I.H.

²: Research Career Development Awardee of the National Institute of General Medical Sciences, N.I.H. (K3-GM-12,638). Present address: Department of Chemistry, University of California, San Diego, La Jolla, California 92037.

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The activation and inactivation of the acyl phosphatase activity of glyceraldehyde-3-phosphate dehydrogenase☆

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