Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase
Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate, and the product was characterized by /sup 31/P, /sup 1/H, and /sup 13/C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by /sup 31/P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 /sup 0/C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by /sup 18/O incorporation from H/sub 2//sup 18/O into P/sub i/, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k/sub cat/ values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.
- Research Organization:
- Univ. of Wisconsin, Madison
- OSTI ID:
- 5876637
- Journal Information:
- Biochemistry; (United States), Vol. 26:13
- Country of Publication:
- United States
- Language:
- English
Similar Records
Mechanism of N[superscript 10]-formyltetrahydrofolate synthetase derived from complexes with intermediates and inhibitors
/sup 18/O isotope effect in /sup 13/C nuclear magnetic resonance spectroscopy. Part 9. Hydrolysis of benzyl phosphate by phosphatase enzymes and in acidic aqueous solutions
Related Subjects
LIGASES
BIOCHEMICAL REACTION KINETICS
PHOSPHORIC ACID ESTERS
CHEMICAL ACTIVATION
NUCLEAR MAGNETIC RESONANCE
SUBSTRATES
ADP
CARBON 13
CLOSTRIDIUM
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
FOLIC ACID
LABELLED COMPOUNDS
MAGNESIUM COMPOUNDS
OXYGEN 18
PH VALUE
PHOSPHORUS 31
PROTONS
ALKALINE EARTH METAL COMPOUNDS
AMINO ACIDS
AROMATICS
AZAARENES
BACTERIA
BARYONS
CARBON ISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
DRUGS
ELEMENTARY PARTICLES
ENZYMES
ESTERS
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROLYSIS
HYDROXY COMPOUNDS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYSIS
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEI
NUCLEONS
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXYGEN ISOTOPES
PHOSPHORUS ISOTOPES
PTERIDINES
REACTION KINETICS
RESONANCE
SOLVOLYSIS
STABLE ISOTOPES
VITAMIN B GROUP
VITAMINS
550601* - Medicine- Unsealed Radionuclides in Diagnostics