Summary
The naturally occurring pteroylpolyglutamate derivatives are substrates for the folate-mediated reactions in cells, including the reactions catalyzed by two multifunctional folate dependent enzymes in eucaryotes. The appropriate derivatives of tetrahydropteroyl (glutamate)n where n = 1, 3, 5, or 7 were used to determine the specificity for, and kinetic advantages of the extra glutamyl residues with two multifunctional proteins from pig liver: methylene-tetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, and formiminotransferase-formimotetrahydrofolate cyclodeaminase. Specificity for the polyglutamate derivatives ranged from 10-to 70-fold as indicated from Km values or from the ability to inhibit the five different enzyme activities. With the sequential activities of the transferase-deaminase enzyme, it was demonstrated that when the tetrahydropteroyl pentaglutamate is used as a substrate, the intermediate formimino-compound does not accumulate in the medium. That this kinetic observation is due to preferential transfer of the pentaglutamate- but not monoglutamate intermediate from transferase to deaminase sites without its release from the enzyme molecule was supported by three types of experiments. Chemical modification to yield monofunctional derivatives of the transferase-deaminase affected the kinetics of the recombined activities only with the pentaglutamate substrate, causing a lag in the appearance of final product. Inhibition studies demonstrated that the deaminase activity could preferentially be inhibited only with the monoglutamate substrate. The deaminase activity with the monoglutamate substrate was increased by providing elevated formiminotetrahydrofolate in the assay mixture; no effect was observed when the reaction was carried out with pentaglutamate. Preliminary binding studies indicate a single folate site per subunit of the octameric enzyme, suggesting a type of combined transferase-deaminase site.
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MacKenzie, R.E., Baugh, C.M. (1983). Interaction of Tetrahydropteroylpolyglutamates with Two Folate-Dependent Multifunctional Enzymes. In: Goldman, I.D., Chabner, B.A., Bertino, J.R. (eds) Folyl and Antifolyl Polyglutamates. Advances in Experimental Medicine and Biology, vol 163. Springer, Boston, MA. https://doi.org/10.1007/978-1-4757-5241-0_2
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