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Abstract
The yeast ADE3 gene encodes the trifunctional protein C(,1)-THF synthase. C(,1)-THF synthase interconverts one carbon folate derivatives that are used in nucleotide, amino acid, vitamin, and protein biosynthesis. The three enzyme activities of C(,1)-THF synthase are on one trifunctional protein in the eukaryotes, but no corresponding trifunctional protein has been purified from prokaryotes. I have characterized the yeast C(,1)-THF synthase in an effort to understand the trifunctional nature of this protein and to compare this trifunctional protein to the proteins of prokaryotes which are organized differently.
An improved purification of yeast C(,1)-THF synthase has simplified the preparation of the enzyme for physical studies. The kinetic mechanism of the synthetase activity of the yeast protein is a random sequential mechanism similar to the mechanism of the prokaryotic 10-CHO-THF synthetases. Antibodies to the yeast protein react with the trifunctional C(,1)-THF synthases and the prokaryotic 10-CHO-THF synthetases. Antibody to the Clostridium acidi-urici synthetase also reacts with C(,1)-THF synthases. These cross-reactions indicate that these proteins, though widely diverged in structure, share some structural feature. To more closely investigate the structure of the yeast protein, the ADE3 gene encoding it was sequenced. The sequences of the ADE3 gene and the protein's N-terminus prove that C(,1)-THF synthase is the ADE3 gene product. A structural model in which the dehydrogenase and cyclohydrolase activities are localized to the N-terminal domain and the synthetase to the C-terminus was proposed from the sequence. This sequence provides detailed structural information for an investigation of the relationship of structure to function for trifunctional C(,1)-THF synthase.
