Abstract
The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl. Reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or methoxycarbonyl coenzyme A (CoA) disulfide, leads to a total loss of enzyme activity. DTNB inactivation is biphasic (k1 = approximately 9 x 10(2) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1) with the slow phase being diminished by acetyl-CoA. Methoxycarbonyl-CoA disulfide inactivation is also biphasic (k1 = approximately 2.1 x 10(3) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1), with the rapid phase being diminished in the presence of acetyl-CoA. Inactivation by methoxycarbonyl methyl disulfide, ethyl disulfide, or hydroxyethyl disulfide, or by methyl methanethiosulfonate is not biphasic. Pretreatment of the enzyme with methyl methanethiosulfonate, which leads to a 25% loss in enzyme activity, abolishes the fast phase of DTNB inactivation, the slow phase of methoxycarbonyl-CoA disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity.
Highlights
The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl
Disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity
Methoxycarbonyl alkyl disulfides have with protein sulfhydryl groups according which protein-alkyl disulfides are formed
Summary
Enzyme Assays-All enzyme assays were conducted at 37°C employing the fluorometric assay previously described [13]. ATP-citrate lyase employed in the assay system was not inactivated at an appreciable rate This was routinely determined by testing the integrity of the coupling system in the same sample in which the activity of choline acetyltransferase had been determined previously. NMR spectra were obtained for both CoA and the product, methoxycarbonyl-CoA disulfide, in deuterium oxide. Methoxycarbonyl-CoA disulfide exhibited essentially the same NMR spectrum as CoA, with the exception that a single new peak at 3.78 6 was obtained with this compound. The chemical shift and integration of this new peak indicated the presence of the 3 protons expected from the methoxy group of methoxycarbonyl-CoA disulfide. The compound contained no free CoA as judged by its inability to react with DTNB or to serve as a substrate in the ATP citrate lyase reaction. After reaction with 20 mM dithiothreitol for 30 min at room temperature, CoA was quantitatively liberated as measured by reaction with ATP citrate lyase
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