title: Purification and properties of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from L1210 cells.
abstract: Serine hydroxymethyltransferase and the trifunctional enzyme C1-tetrahydrofolate synthase have been purified to near homogeneity from L1210 cells. Kinetic constants (Km and kcat) have been determined for both folate and non-folate substrates. The effect of increasing glutamate chain length on affinity and catalytic efficiency were determined for the four activities. The studies show that the structural and catalytic properties of the two L1210 enzymes are very similar to the corresponding enzymes purified from rabbit liver. Antibodies to both rabbit serine hydroxymethyltransferase and C1-tetrahydrofolate synthase cross-react with the corresponding L1210 enzymes. The intracellular concentration of active sites of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in L1210 cells are both 9 microM. The combined concentration of these two enzymes exceeds the previously reported concentration of 10 microM for total intracellular folates. A network thermodynamic computer model of one carbon metabolism (Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. (1989) J. Biol. Chem. 264, 17016-17023) suggests that complete inhibition of cytosolic serine hydroxymethyltransferase would neither significantly decrease the rates of biosynthesis of purines and thymidylate nor significantly alter the rate of interconversion of tetrahydrofolate cofactors to dihydrofolate with subsequent inhibition of dihydrofolate reductase.
authors.affiliation: Department of Biochemistry, Virginia Commonwealth University Medical College of Virginia, Richmond 23298.
authors.forename: W B
authors.initials: WB
authors.lastname: Strong
authors.forename: S J
authors.initials: SJ
authors.lastname: Tendler
authors.forename: R L
authors.initials: RL
authors.lastname: Seither
authors.forename: I D
authors.initials: ID
authors.lastname: Goldman
authors.forename: V
authors.initials: V
authors.lastname: Schirch
mesh_terms: D000619:Aminohydrolases / Q000302:isolation & purification*; D000818:Animals; D002845:Chromatography; D005574:Formate-Tetrahydrofolate Ligase / Q000302:isolation & purification*; D012696:Glycine Hydroxymethyltransferase / Q000302:isolation & purification*; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology; D008025:Ligases / Q000302:isolation & purification*; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000302:isolation & purification*; D051379:Mice; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D010088:Oxidoreductases / Q000302:isolation & purification*; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D014166:Transferases / Q000302:isolation & purification*
chemical_list: D009097:Multienzyme Complexes; D011624:Pteroylpolyglutamic Acids; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D014166:Transferases; D012696:Glycine Hydroxymethyltransferase; D000619:Aminohydrolases; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase