Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase

Bloxham, David P.; Coghlin, S J; Sharma, Ram Prakash

doi:10.1016/0005-2744(78)90200-0

Cited by 15 publications

(9 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[3H]Methyl methanethiosulphonate The radioactive compound was synthesized by the condensation of [3H]methyl iodide with potassium methylthiosulphonate (Bloxham et al, 1978). It was finally obtained by vacuum-distillation.…”

Section: Reduction Ofthio[3h]methylated Lactate Dehydrogenasementioning

confidence: 99%

“…Potassium methanethiosulphonate (3.0g; 0.02mol) was prepared as described by Bloxham et al (1978) and dissolved in dry NN-dimethylformamide (15 ml). Ethyl iodide (4.5g; excess) was added, and the reaction mixture was stirred at room temperature for 5 h. Water (100ml) was then added to the reaction mixture and it was extracted with chloroform (3 x 50ml).…”

Section: Synthesis Of Ethyl Methanethiosulphonatementioning

confidence: 99%

“…We have been using the reagent methyl methanethiosuphonate to thiomethylate proteins, in order to investigate the role of thiol groups in catalysis (Bloxham & Wilton, 1977;Bloxham et al, 1978). Pig heart lactate dehydrogenase is one of the proteins that is susceptible to thiomethylation and this leads to an enzyme with altered catalytic properties compared with the native enzyme (Bloxham & Wilton, 1977).…”

mentioning

confidence: 99%

See 2 more Smart Citations

A detailed investigation of the properties of lactate dehydrogenase in which the ‘Essential’ cysteine-165 is modified by thioalkylation

Bloxham¹,

Sharma²,

Wilton³

1979

Biochemical Journal

View full text Add to dashboard Cite

The reaction of pig heart lactate dehydrogenase with methyl methanethiosulphonate resulted in the modification of one thiol group per protomer, and this was located at cysteine-165 in the enzyme sequence. On reduction, both the thiomethylation of cysteine-165 and any changes in kinetic properties of the enzyme were completely reversed. Cysteine-165 has been considered essential for catalytic activity; however, cysteine-165-thiomethylated dehydrogenase possessed full catalytic activity, although the affinity of the enzyme for carbonyl-or hydroxy-containing substrates was markedly decreased. The nicotinamide nucleotide-binding capacity was unaffected, as judged by the formation of fluorescent complexes with NADH. The enzyme-mediated activation of NAD+, as judged by sulphite addition, was unaffected in thiomethylated lactate dehydrogenase. However, the affinity of oxamate for the enzyme--NADH complex was decreased by 100-fold and it was calculated that this constituted a net increase of 10.4 kJ/mol in the activation energy for binding. Thiomethylated lactate dehydrogenase was able to form an abortive adduct between NAD+ and fluoropyruvate. However, the equilibrium constant for adduct formation between pyruvate and NAD+ was too low to demonstrate this complex at reasonable pyruvate concentrations. A conformational change in the protein structure on selective thiomethylation was revealed by the decreased thermostability of the modified enzyme. The alteration of lactate dehydrogenase catalytic properties on modification depended on the bulk of the reagent used, since thioethylation resulted in an increase in Km for pyruvate (13.5 +/- 3.5 mm) and an 85% decrease in maximum catalytic activity. The implications of all these findings for the catalytic mechanism of lactate dehydrogenase are discussed.

show abstract

Section: Reduction Ofthio[3h]methylated Lactate Dehydrogenasementioning

confidence: 99%

Section: Synthesis Of Ethyl Methanethiosulphonatementioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

A detailed investigation of the properties of lactate dehydrogenase in which the ‘Essential’ cysteine-165 is modified by thioalkylation

Bloxham¹,

Sharma²,

Wilton³

1979

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…were synthesized by condensation, in refluxing methanol, between a dibromoalkane and an excess of potassium methanethiosulphonate (Scheme 1). Reactions of this type are generally performed in NNdimethylformamide (Smith et al, 1975;Bloxham et al, 1978;Corina et al, 1979), but methanol was preferred in these syntheses because, although the reaction rate is considerably lower, the isolation of product is considerably easier. ]_3PB3r33,C disulphide (Scheme 1).…”

Section: Resultsmentioning

confidence: 99%

“…Synthesis ofa series ofSS '-polymethylenebis(methanethiosulphonates) Potassium methanethiosulphonate was prepared as described by Bloxham et al (1978). First 2.5 mmol of dibromoalkane was gently refluxed with potassium methanethiosulphonate (6.6 mmol) in 13 ml of dry methanol for 18 h. Solvent was then removed in vacuo and the solid was mixed with 100ml of warm chloroform to dissolve the product.…”

Section: Methodsmentioning

confidence: 99%

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

Bloxham¹,

Sharma²

1979

Biochemical Journal

View full text Add to dashboard Cite

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.

show abstract

Chemical ionization and electron impact mass spectra of symmetrical S‐alkyl alkanesulfonothioates (thiosulfonates)

Freeman¹,

Keindl²

1992

Org. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase

Cited by 15 publications

References 33 publications

A detailed investigation of the properties of lactate dehydrogenase in which the ‘Essential’ cysteine-165 is modified by thioalkylation

A detailed investigation of the properties of lactate dehydrogenase in which the ‘Essential’ cysteine-165 is modified by thioalkylation

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

Chemical ionization and electron impact mass spectra of symmetrical S‐alkyl alkanesulfonothioates (thiosulfonates)

Contact Info

Product

Resources

About