SH Groups Of Enzyme Research Articles

Participation of SH groups in the auto-activation of insect dihydrofolate reductase

1. 1. Some extraneous proteins retarded the auto-activation of the insect dihydrofolate reductase. Their effect seems to be connected with protection of the enzyme SH groups. 2. 2. All tested thiol compounds prevented and reversed the auto-activation of dihydrofolate reductase. 3. 3. The enzyme was found to be activated by several SH reagents and their effect was also prevented and reversed by thiol compounds. 4. 4. The supposition was made that during the process of auto-activation of the insect dihydrofolate reductase, oxidation of its SH groups occurs, which results in a catalytically more favourable enzyme conformation.

L'ATP: Thiamine pyrophosphotransférase. Purification et étude du mécanisme de réaction

ATP:thiamine pyrophosphotransferase. Purification and reaction mechanism study 1. 1. ATP:thiamine pyrophosphotransferase (EC 2.7.6.2) from baker's yeast has been purified 200-fold. 2. 2. The enzyme SH groups are not very reactive. The kinase is not completely inhibited by p-chloromercuribenzoate, N-ethylmaleimide and 5,5′-dithio-bis-z-nitrobenzoic acid even at high concentrations. Iodoacetic acid and iodoacetamide are unreactive. 3. 3. The enzyme is fully inhibited by 2,4,6-trinitrobenzene sulfonic acid and strongly by 5-dimethylaminonaphthalane sulfonyl chloride and by maleic anhydride. 4. 4. Some kinetic aspects of the reaction have been examined. The double reciprocal plots of initial velocity versus thiamine concentration at different levels of ATP concentration give a family of parallel lines. In agreement with this result, plotting i/velocity versus i/concentration of ATP for some different ATP/thiamine ratios yields straight lines intercepting on the ordinate axis at the same point. The results of kinetic studies are consistent with a “ping-pong” reaction mechanism.

X-ray modification of the catalytic and allosteric functions of fructose-1,6-diphosphatase

1. 1. Native rabbit liver fructose-1,6-diphosphatase ( d-fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11) and enzyme stimulated by disulfide formation have been irradiated in solution with X-rays. 2. 2. The dose-response curve for the native enzyme showed an initial stimulation of catalytic activity, followed by subsequent exponential inactivation. The disulfide-modified enzyme was inactivated exponentially with no initial stimulation. The G values for inactivation of catalytic activity were 0.017 and 0.009 for the native and the disulfide-modified enzymes, respectively. 3. 3. The allosteric activity of the native enzyme, as measured by the inhibition by AMP, was much more radioresistant than the catalytic activity. In the modified enzyme, these two functions were of comparable radiosensitivity. 4. 4. The ability of the enzymes to be stimulated by mixed disulfide formation was about 10 times as sensitive to X-ray inactivation as was the catalytic function. The loss of sensitivity to stimulation by disulfides was due to destruction of 5–6 specific SH groups which were particularly radiosensitive ( G value, 1.2). 5. 5. The X-ray-induced reduction in enzyme activity was associated with destruction of enzyme SH groups of low radiosensitivity ( G value, 0.07. The loss of enzyme activity was partly reversed by addition of cysteamine after exposure. The results indicate that sulfhydryl groups play an important, but not exclusive role in the X-ray inactivation of fructose-1,6-diphosphatase. 6. 6. The presence of AMP during irradiation protected the enzyme almost completely against inactivation of the allosteric function. The results indicated that the X-ray inactivation of the allosteric function of fructose-1,6-diphosphatase is due to a specific modification of the binding sites for AMP.

Stimulation of dihydrofolate reductase activity by [formula omitted]-chloromercuribenzoate

1.1. Some extraneous proteins retarded the auto-activation of the insect dihydrofolate reductase. Their effect seems to be connected with protection of the enzyme SH groups.2.2. All tested thiol compounds prevented and reversed the auto-activation of dihydrofolate reductase.3.3. The enzyme was found to be activated by several SH reagents and their effect was also prevented and reversed by thiol compounds.4.4. The supposition was made that during the process of auto-activation of the insect dihydrofolate reductase, oxidation of its SH groups occurs, which results in a catalytically more favourable enzyme conformation.

The radiosensitizing effect of thioglycolic acid, dithiodiglycolic acid, and homocystine on muscle glyceraldehyde-3-phosphate dehydrogenase.

SummaryIt is demonstrated that thioglycolic acid and its disulphide, dithiodiglycolic acid, as well as homocystine, have a pronounced radiosensitizing effect on the x-ray inactivation of crystalline rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase in solution. A number of other thiols and disulphides tested exert a protective effect.The radiosensitization observed is associated with a disappearance of free enzyme SH-groups, and it can be reversed by addition of excess thiols after the radiation exposure. The data indicate that the sensitizing mechanism involves a blocking of essential enzyme SH-groups through a radiochemical formation of mixed disulphides between the enzyme SH-groups and the radiosensitizing substances.

The Mechanism of X-ray Inactivation of Phosphoglyceraldehyde Dehydrogenase

SummaryThe x-ray inactivation of crystalline rabbit-muscle phosphoglyceraldehyde dehydrogenase (GAPDH) in solution has been correlated with the concurrent radiochemical destruction of enzyme SH-groups. The inactivation was found to be directly proportional to the disappearance of enzyme SH-groups and was complete when three SH-groups had been destroyed. Exactly the same relationship between enzyme inactivation and disappearance of free SH-groups was found when an increasing number of SH-groups were blocked by the addition of p-chloromercuribenzoate. The data strongly indicate that the x-ray inactivation of GAPDH can be entirely accounted for by destruction of its SH-groups.The G-value for the x-ray destruction of the enzyme SH-groups was found to be 0·23, indicating that the major part of the radiation energy is dissipated in processes not involving the protein SH-groups. Blocking of enzyme SH-groups by p-chloromercuribenzoate prior to the irradiation provided partial protection of the enzyme. The data are consistent with the view that, contrary to current assumptions, all SH-groups of GAPDH are of importance for the enzyme activity.

The effect of oxygen on the growth and metabolism of the aerobic thermophilic actinomycete Micromonospora vulgaris.

SUMMARY: In the presence of pure oxygen at one atmosphere pressure Micromonospora vulgaris is unable to form in liquid media a surface pellicle of growth-bearing aerial mycelium. The production of bottom growth is either unaffected or stimulated by oxygen. Vegetative mycelium which develops from spores in Warburg vessels has an oxygen uptake that is similar in the presence of air or oxygen. Aerial mycelium, harvested from growths in air. shows marked differences as regards oxygen uptake in the presence of air or oxygen; oxygen is inhibitory. Some evidence is presented to show that the effect of oxygen may be connected with the inactivation of essential -SH groups of thiol enzymes.