Co-purification of dihydrofolate synthetase and N/sub 10/formyltetrahydropteroyldiglutamate synthetase from E. coli

Journal Article · Sat Jan 01 00:00:00 EST 1983 · Adv. Exp. Med. Biol.; (United States)

DOI:https://doi.org/10.1007/978-1-4757-5241-0_14· OSTI ID:5552038

Ferone, R; Warskow, A

The enzymatic activities which add a single glutamate to dihydropteroate (H/sub 2/Pte) and to N/sub 10/formyltetrahydropteroylglutamate (N/sub 10/formylH/sub 4/PteGlu) remained at a constant ratio when various purification techniques were used, including ammonium sulfate fractionation, isoelectric focusing, polyacrylamide gel electrophoresis, and column chromatography on Sephadex G-100, DEAE-Sephadex, CM-Sephadex, ADP-Sepharose, N/sub 10/formylfolate-agarose or matrix Gel Red-A. The best combination of these methods yielded 900-fold purified enzyme. However, the kinetic properties were dependent upon the substrate used. H/sub 2/Pte was a noncompetitive inhibitor (Kii . 1.1 microM) of the utilization of N/sub 10/formylH/sub 4/PteGlu, but no inhibition was detected in the reciprocal experiment. Aminopterin was a competitive inhibitor (Kis . 370 microM) of the reaction with N/sub 10/formylH/sub 4/PteGlu but was not inhibitory with H/sub 2/Pte as substrate. In the dihydrofolate synthetase reaction, in Tris-HCl, pH 8.9 with 50 mM KCl, the apparent Km values for glutamate and MgATP were 3.5 mM and 8.1 microM respectively. With N/sub 10/formylH/sub 4/PteGlu as substrate, these Km values were 1.2 mM and 80 microM. Since the two activities were not separated by protein purification procedures but exhibited different kinetic properties (including lack of reciprocal inhibition), the data suggest these reactions are catalyzed on independent sites of a common protein.

• Research Organization: Department of Microbiology, Wellcome Research Laboratories, Research Triangle Park, North Carolina
• OSTI ID: 5552038
• Journal Information: Adv. Exp. Med. Biol.; (United States), Vol. 163
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LIGASES
BIOCHEMICAL REACTION KINETICS
INHIBITION
PURIFICATION
PROTEINS
ELECTROPHORESIS
LIQUID COLUMN CHROMATOGRAPHY
ATP
ENZYME INHIBITORS
PTERIDINES
AROMATICS
AZAARENES
CHROMATOGRAPHY
ENZYMES
HETEROCYCLIC COMPOUNDS
KINETICS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
REACTION KINETICS
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques