Multiple oxidation products of sulfhydryl groups near the active site of thiolase I from porcine heart

Biochemistry ◽  
1984 ◽  
Vol 23 (19) ◽  
pp. 4318-4324 ◽  
Author(s):  
Elzbieta Izbicka-Dimitrijevic ◽  
Hiram F. Gilbert
Keyword(s):  
Active Site ◽  
Sulfhydryl Groups ◽  
Oxidation Products ◽  
Porcine Heart

Influence of Oxidative Stress on Catalytic and Non-glycolytic Functions of Glyceraldehyde-3-phosphate Dehydrogenase

2020 ◽  
Vol 27 (13) ◽  
pp. 2040-2058 ◽  
Author(s):  
Vladimir I. Muronetz ◽  
Aleksandra K. Melnikova ◽  
Luciano Saso ◽  
Elena V. Schmalhausen
Keyword(s):  
Oxidative Stress ◽  
Disulfide Bond ◽  
Active Site ◽  
Sulfhydryl Groups ◽  
Oxidation Products ◽  
Main Function ◽  
Complete Inactivation ◽  
Mixed Disulfide ◽  
Oxidation Reduction ◽  
Catalytic Cysteine

Background: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) is a unique enzyme that, besides its main function in glycolysis (catalysis of glyceraldehyde-3-phosphate oxidation), possesses a number of non-glycolytic activities. The present review summarizes information on the role of oxidative stress in the regulation of the enzymatic activity as well as non-glycolytic functions of GAPDH. Methods: Based on the analysis of literature data and the results obtained in our research group, mechanisms of the regulation of GAPDH functions through the oxidation of the sulfhydryl groups in the active site of the enzyme have been suggested. Results: Mechanism of GAPDH oxidation includes consecutive oxidation of the catalytic Cysteine (Cys150) into sulfenic, sulfinic, and sulfonic acid derivatives, resulting in the complete inactivation of the enzyme. The cysteine sulfenic acid reacts with reduced glutathione (GSH) to form a mixed disulfide (S-glutathionylated GAPDH) that further reacts with Cys154 yielding the disulfide bond in the active site of the enzyme. In contrast to the sulfinic and sulfonic acids, the mixed disulfide and the intramolecular disulfide bond are reversible oxidation products that can be reduced in the presence of GSH or thioredoxin. Conclusion: Oxidation of sulfhydryl groups in the active site of GAPDH is unavoidable due to the enhanced reactivity of Cys150. The irreversible oxidation of Cys150 is prevented by Sglutathionylation and disulfide bonding with Cys154. The oxidation/reduction of the sulfhydryl groups in the active site of GAPDH can be used for regulation of glycolysis and numerous side activities of this enzyme including the induction of apoptosis.

scholarly journals Structure-Function Analysis Indicates that an Active-Site Water Molecule Participates in Dimethylsulfoniopropionate Cleavage by DddK

2019 ◽  
Vol 85 (8) ◽  
Author(s):  
Ming Peng ◽  
Xiu-Lan Chen ◽  
Dian Zhang ◽  
Xiu-Juan Wang ◽  
Ning Wang ◽  
...  
Keyword(s):  
Water Molecule ◽  
Active Site ◽  
Dimethyl Sulfide ◽  
Catalytic Mechanism ◽  
Heterotrophic Bacteria ◽  
Function Analysis ◽  
Cloud Condensation Nuclei ◽  
Oxidation Products ◽  
Condensation Nuclei ◽  
Marine Heterotrophic Bacteria

ABSTRACT The osmolyte dimethylsulfoniopropionate (DMSP) is produced in petagram quantities in marine environments and has important roles in global sulfur and carbon cycling. Many marine microorganisms catabolize DMSP via DMSP lyases, generating the climate-active gas dimethyl sulfide (DMS). DMS oxidation products participate in forming cloud condensation nuclei and, thus, may influence weather and climate. SAR11 bacteria are the most abundant marine heterotrophic bacteria; many of them contain the DMSP lyase DddK, and their dddK transcripts are relatively abundant in seawater. In a recently described catalytic mechanism for DddK, Tyr64 is predicted to act as the catalytic base initiating the β-elimination reaction of DMSP. Tyr64 was proposed to be deprotonated by coordination to the metal cofactor or its neighboring His96. To further probe this mechanism, we purified and characterized the DddK protein from Pelagibacter ubique strain HTCC1062 and determined the crystal structures of wild-type DddK and its Y64A and Y122A mutants (bearing a change of Y to A at position 64 or 122, respectively), where the Y122A mutant is complexed with DMSP. The structural and mutational analyses largely support the catalytic role of Tyr64, but not the method of its deprotonation. Our data indicate that an active water molecule in the active site of DddK plays an important role in the deprotonation of Tyr64 and that this is far more likely than coordination to the metal or His96. Sequence alignment and phylogenetic analysis suggest that the proposed catalytic mechanism of DddK has universal significance. Our results provide new mechanistic insights into DddK and enrich our understanding of DMS generation by SAR11 bacteria. IMPORTANCE The climate-active gas dimethyl sulfide (DMS) plays an important role in global sulfur cycling and atmospheric chemistry. DMS is mainly produced through the bacterial cleavage of marine dimethylsulfoniopropionate (DMSP). When released into the atmosphere from the oceans, DMS can be photochemically oxidized into DMSO or sulfate aerosols, which form cloud condensation nuclei that influence the reflectivity of clouds and, thereby, global temperature. SAR11 bacteria are the most abundant marine heterotrophic bacteria, and many of them contain DMSP lyase DddK to cleave DMSP, generating DMS. In this study, based on structural analyses and mutational assays, we revealed the catalytic mechanism of DddK, which has universal significance in SAR11 bacteria. This study provides new insights into the catalytic mechanism of DddK, leading to a better understanding of how SAR11 bacteria generate DMS.

scholarly journals Formation of N-hydroxy-N-arylacetamides from nitroso aromatic compounds by the mammalian pyruvate dehydrogenase complex

1993 ◽  
Vol 290 (3) ◽  
pp. 783-790 ◽  
Author(s):  
T Yoshioka ◽  
T Uematsu
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Aromatic Compounds ◽  
Pyruvate Dehydrogenase Complex ◽  
Catalytic Efficiency ◽  
Nitroso Compounds ◽  
Factors Affecting ◽  
Porcine Heart ◽  
Alkyl Groups ◽  
Dehydrogenase Complex

Bovine, human and porcine heart mitochondria and isolated porcine heart pyruvate dehydrogenase complex (PDHC) pyruvate-dependently form N-hydroxy-N-arylacetamides from nitroso aromatic compounds, including carcinogenic 4-biphenyl and 2-fluorenyl derivatives. The PDHC-catalysed formation of N-hydroxyacetanilide (N-OH-AA) from nitrosobenzene (NOB), through a Ping Pong mechanism, is optimum at pH 6.8 and is accelerated by thiamin pyrophosphate, but is inhibited by thiamin thiazolone pyrophosphate and ATP. Km pyruvate in the reaction is independent of pH over the range tested, whereas KmNOB increases at lower pH, owing to ionization of an active-site functional group of pKa 6.3. The enzymic ionization decreases log (Vmax/KmNOB). Isolated pyruvate dehydrogenase (E1), a constitutive enzyme of PDHC, forms N-OH-AA by itself and has comparable kinetic parameters to those of the PDHC-catalysed N-OH-AA formation. The catalytic efficiency of PDHC in the formation of N-hydroxy-N-arylacylamides, due to the steric limitation of the active site of E1, is lowered both by bulky alkyl groups of alpha-oxo acids and by p-substituents (but not an o-substituent) on nitrosobenzenes. These nitroso compounds serve as electrophiles in the reaction in which the reductive acetylation step is rate-limiting. The reaction mechanism and other factors affecting N-hydroxy-N-arylacylamide formation are discussed.

The Sulfhydryl Groups Involved in the Active Site of Myosin B Adenosinetriphosphatase

1977 ◽  
Vol 82 (4) ◽  
pp. 1085-1092 ◽  
Author(s):  
Tsuneyoshi HORIGOME ◽  
Tatsuhisa YAMASHITA
Keyword(s):  
Active Site ◽  
Sulfhydryl Groups ◽  
Myosin B
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