In vitro phosphorylation of vitamin B12 dependent methionine synthase by protein kinase A.

Abstract

Methioninesynthase( L-homocysteine-S-methyltransferase) is a cytosolic enzyme catalysing the formation of methionine and tetrahydrofolate from homocysteine and methyltetrahydrofolate. It is one of two vitamin B,, (cobalamin) dependent enzymes, in mammals, the other being methylmalonyl CoA mutase. In bacteria a cobalamin independent form of methionine synthase also exists’. In vitamin B,, deficiency methionine synthase activity is affected. Symptoms of vitamin B,, deficiency include: pernicious anaemia, neurological and psychiatric abnormalities*. Inhibition of methionine synthase is considered to be important in producing, at least some, of these symptoms as it stands at the convergence point of the folate metabolic pathway as well as the sulphur amino acid pathway. Inactivation of methionine synthase will thus affect folate metabolism, methionine, polyamine & S-adenosylmethionine production (and hence methylation reactions) and homocysteine metabolism’. I t can be seen that methionine synthase is biologically important. So it was of interest to us to study the regulation of this enzyme. There are a couple of reports about the regulation of gene transcription in bacteria4. I t has also been suggested that enzyme activity could be regulated by controlling the avaliability of cobalamin. This seems unlikely as most of the body’s vitamin B,, is stored in the enzyme bound form’. Nitric oxide has been shown to inhibit methionine synthase, although it is uncertain whether this could be a physiological or toxic effect6. A logical first step to initiate studies on possible mechanisms for regulating methionine synthase was to ascertain whether protein kinases changed the activity of the enzyme, as kinases are well known regulators of many enzymatic processes. Here we report preliminary investigations into the possible regulation of methionine synthase by protein kinase A. Methionine synthase activity was assayed for, using a modified version of the method of Weisbech et al’ and incubating for various times up to 30 mins. Protein kinase A, 5 picounits (from Sigma 1.3 pmolar units is equivalent to Ipg) was introduced into the standard assay mixture, along with ImM ATP, 2.2mM MgClz and 12.6pM CAMP. Control assays contained all elements except the kinase, to account for the magnesium induced stimulation of the methionine synthase. A time dependent study was performed and can be seen in figure I . The experiment was performed on two methionine synthase samples at different stages of purification6. The purer sample showed a four fold increase in stimulation (table I ) . I 1 I I I I I Sample