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Biochemistry. 1987 Jan 27;26(2):513-21.

Mammalian folylpoly-gamma-glutamate synthetase. 2. Substrate specificity and kinetic properties.

Abstract

The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and 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 (kcat = 2.5 s-1) while 5- and 10-position substitutions of the folate molecule impair catalysis. kcat 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. The on rates for most pteroyldiglutamates are similar to the rates for their respective monoglutamate derivatives, but further extension of the glutamate chain results in a progressive decrease in on rates. 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 Km 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), P1,P5-di(adenosine-5') pentaphosphate, and free ATP4- are potent inhibitors of the reaction.

PMID:
3828321
DOI:
10.1021/bi00376a025
[Indexed for MEDLINE]

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