Graduate Thesis Or Dissertation
 

The terminal reactions in the biosynthesis of methionine in Saccharomyces cerevisiae

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  • The terminal reactions in the biosynthetic pathway of methionine in Saccharomyces cerevisiae were investigated in this study. Analyses of a number of methionine auxotrophs for biochemical deficiences established four mutant groups. It was observed from qualitative feeding experiments that some auxotrophs were able to utilize S-adenosylmethionine (AM) and S-adenosyl-homocysteine (AH) to satisfy their methionine requirement indicating a possible role of these. compounds in the biosynthetic pathway. Enzymatic analyses indicated further, differences in the mutant groups. One group, designated as Me-1, was unable to substitute any suspected intermediate tested for the methionine requirement. These organisms also lacked any S-adenosylmethionine-homocysteine transmethylase activity. The mutant group designated as Me-2 included organisms which were able to utilize S-adenosylmethionine or methionine for growth; this group was further divided following completion of serine hydroxymethylase assays with those organisms showing no activity of this enzyme now noted as the Me-5 group. Methionine synthesis was accomplished in cell-free preparations of Saccharomyces cerevisiae utilizing serine as the one-carbon donor. Synthesis was obtained using a boiled enzymatic extract or a Sephadex column eluate of an enzymatic extract. To determine the natural cofactor involved a number of suspected cofactors were tested in the system. Methyl tetrahydrofolate, methyl B₁₂, tetrahydrofolate, and dihydrofolate inhibited methionine synthesis. However, Teropterin stimulated activity indicating that Teropterin may be the cofactor involved or may be converted to the natural cofactor by our cell-free preparation. The synthesis of methionine was dependent upon the availability of S-adenosyl-homocysteine indicating that this compound may be methylated to S-adenosylmethionine in this pathway. It seems evident that S-adenosylmethionine can be formed in yeast in two ways: by the direct methylation of S-adenosyl-homocysteine or by the reaction of methionine and adenosine triphosphate catalyzed by AM synthetase. A study of AM synthetase was also performed, and it was shown that AM synthetase is inducible in the presence of large concentrations of methionine. Recent reports indicate that in Escherichia coli two independent systems for methionine biosynthesis are functional, one being dependent upon vitamin B₁₂ and the other independent of vitamin B₁₂. Comparative studies indicate that in mammalian systems vitamin B₁₂ and folic acid derivatives are also involved.The data obtained in this study suggest that although the terminal reactions of methionine biosynthesis in Saccharomyces cerevisiae have characteristics of both the vitamin B₁₂-dependent and vitamin B₁₂ independent systems, the yeast pathway is unique. A scheme which is compatible with the data is suggested; the terminal reactions would involve at least three enzymes including serine hydroxymethylase, S-adenosyl-homocysteine methylase and S-adenosylmeth ionise-homocysteine transmethylase with Teropterin or a product of Teropterin, S-adenosyl-homocysteine and S-adenosylmethionine serving as intermediates.
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