This study seeks to determine properties of a major enzyme of liver, glycine N-methyltransferase (GNMT). We had discovered that GNMT from rat liver binds and is inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-THF-Gs). This is part of a regulatory scheme that links the de novo synthesis of methyl groups via the folate one-carbon pool to the availability of methyl groups from methionine. Recombinant GNMT expressed in E. coil is not inhibited by 5-CH3-THF-Gs. We found that inhibition of native GNMT by 5-CH3-THF-G5 depends upon the order of addition of inhibitor and substrates to the enzyme. Our first hypothesis is that inhibition depends on a post-translational modification not present in the recombinant enzyme. Our second hypothesis is that this post-translational modification may be phosphorylation of the native enzyme. Our third hypothesis is that there are structural and conformational differences between the native and recombinant enzymes. Our fourth hypothesis is that conformational changes in the enzyme take place when enzyme and substrates bind. To test these hypotheses, our first specific aim is to express GNMT transfected into human H1299 cells that carry out post-translational modification and examine inhibition and binding by 5-CH3-THF-Gs. Our second specific aim is to identify the sites of phosphorylation in native rat GNMT, GNMT expressed in H1299 cells and to mutate the phosphorylated amino acids from H1299 cells to determine which are needed for inhibition. Native GNMT is N-terminal acetylated. Our third specific aim is to remove the N-terminal acetyl group to determine whether this the source of the difference between native and recombinant enzyme. Our fourth specific aim is to determine where the inhibitor, 5-CH3-THF-Gs, is bound to the GNMT expressed in H1299. We will do this by solving the crystal structure of GNMT without and with the bound inhibitor. We will also attempt to covalently link 5-CH3-THF-G to GNMT and determine the position of the inhibitor by tryptic digestion and mass spectrometrometry. Our fifth specific aim is to measure conformational changes of native GNMT in the presence of substrates and inhibitor. We will use labeling of GNMT by [32P]-phosphate followed by tryptic digestion, immunopurification of GNMT, HPLC isolation of labeled peptides and Edman sequencing to locate phospho-amino acids as well as mass-spectroscopic identification of phospho-amino acids and any other post-translational modifications. Changes in conformation will be monitored by intrinsic protein fluorescence. Bindin 9 of inhibitor will be measured by ultrafiltration.
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