Mammalian folylpoly-. gamma. -glutamate synthetase. 2. Substrate specificity and kinetic properties
The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and L-(/sup 14/C)glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis while 5- and 10-position substitutions of the folate molecule impair catalysis. k/sub cat/ values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The K/sub m/ for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and ..beta..,..gamma..-methylene-ATP, ..beta..,..gamma..-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P/sup 1/,P/sup 5/-di(adenosine-5') pentaphosphate, and free ATP/sup 4 -/ are potent inhibitors of the reaction.
- Research Organization:
- Johns Hopkins Univ., Baltimore, MD
- OSTI ID:
- 6792992
- Journal Information:
- Biochemistry; (United States), Vol. 26:2
- Country of Publication:
- United States
- Language:
- English
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Mammalian folylpoly-. gamma. -glutamate synthetase. 1. Purification and general properties of the hog liver enzyme
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Related Subjects
CARBON 14 COMPOUNDS
TRACER TECHNIQUES
LIGASES
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
ATP
CATALYSIS
FOLIC ACID
GLUTAMIC ACID
NUCLEOTIDES
SUBSTRATES
AMINO ACIDS
AROMATICS
AZAARENES
CARBOXYLIC ACIDS
CHEMISTRY
DRUGS
ENZYMES
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PTERIDINES
REACTION KINETICS
VITAMIN B GROUP
VITAMINS
550201* - Biochemistry- Tracer Techniques