| E. coli serine hydroxymethyltransferase (eSHMT) is a 94-kDa homodimer whose folding pathway has been established. A slow step involving proline isomerization has been suggested to be the rate-determining step. In Chapter 2, the roles of proline isomerization in the folding process of eSHMT is further investigated. Ten proline mutant eSHMTs were cloned, purified, characterized and the refolding rate determined. The results show that three of the five proline residues studied seem to be critical for maintaining the correct conformation and catalytic function of eSHMT.; Described in Chapters 3 and 4 is the second part of this study which aims at elucidating the structural and functional relationship between 10-formyl-tetrahydrofoate dehydrogenase (FTD) and rabbit cytosolic serine hydroxymethyl-transferase (rcSHMT). FTD is very abundant in the liver cells and binds its own product H4PteGlun very tightly. However, the tightly bound H 4PteGlun does not affect the enzyme steady-state kinetic properties, suggesting that a separate H4PteGlun binding site exists on FTD. By cross-linking 10-CHO-5,8-dideazafolate to the FTD catalytic site and using tritiated 5,8-dideazafolate-polyglutamate, we have demonstrated that the tight binding folate site is separate from the catalytic site. The location of the cross-linked 10-CHO-5,8-dideazafolate at the active site was also determined by amino acid sequencing of an isolated tryptic peptide. The possible physiological functions of the H4PteGlun tight binding site are also tested and discussed.; In Chapter 4, the interaction between FTD and rcSHMT and the mechanism of quinonoid formation are investigated through the construction and characterization of twenty mutant rcSHMTs with single mutation at the folate binding site. Five amino acid residues examined seem to be important for the binding of H4PteGlun as well as the transfer of H4PteGlu n from FTD·H4PteGlun to rcSHMT. Our results support the existence of a channeling mechanism for the transfer of H 4PteGlun from FTD to rcSHMT. Also, an additional step, which might involve a conformational change at the folate binding site, has been suggested to be required for the formation of the quinonoid intermediate.; In the last chapter, we have shown that NMR, the tool which has been used in studies of one-carbon metabolism in yeast and N. crassa, is also applicable to for mammalian cells. The number of cells required has been determined and the preparation of samples for NMR analysis established. In addition, the role of cSHMT and mSHMT in terms of their metabolic directions and the flow of C3 of serine in MCF7 cells is investigated. The preliminary results from dilution experiments suggest that C3 of serine is converted to formate in mitochondria and then enters one-carbon metabolism in the cytosol. Also, formate is a preferred one-carbon donor as compared to serine in MCF7 cells. Finally, the development and application of a sensitive and quick assay for measuring homocysteine level in cells and serum are also discussed. |
