Folylpoly-gamma-glutamate synthetase: Kinetics of multiple glutamate ligations.

Abstract

Folylpoly-gamma-glutamate synthetase (FPGS) is an ATP-dependent ligase that catalyzes the formation of the poly-gamma-glutamate chain of folates and anti-folates such as methotrexate and lometrexol(TM). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether a single glutamate residue or multiple glutamate residues are added for each folate binding event. These are the two mechanistic possibilities, distributive or processive respectively, for successive glutamate additions to the folate substrate. In a distributive mechanism, the enzyme and products dissociate between each addition and the enzyme then randomly distributes itself among all available termini. A processive mechanism is one in which the enzyme processes along the same substrate chain catalyzing multiple additions without dissociating. This research is focused on determining which of these mechanisms is utilized by FPGS and developing methods for the study of multiple ligation reactions. Specifically, recombinant human FPGS (hFPGS) with the antifolate drug lometrexol(TM) were chosen as the enzyme-substrate pair. Two assays have been developed for studying hFPGS. The first is a continuous spectrophotometric assay utilizing the pyruvate kinase/lactate dehydrogenase enzymatic coupling system, used in the study of an ATPase reaction catalyzed by hFPGS. The second assay is a non-continuous, radiation-based assay that relies on ion-pair HPLC separation of the folate products of the hFPGS reaction followed by liquid scintillation counting. A new synthesis of lometrexol(TM) ((6<italic>R </italic>)-5,10-dideazatetrahydrofolic acid), radio-labeled lometrexol(TM), and the poly-gamma-glutamates of lometrexol(TM) for use in the study of the hFPGS reaction is reported. These tools allowed a careful analysis of the time course of formation of the various chain-length products to be accomplished. These time course data were used to determine the relative rates of extension of the different polyglutamate substrates. Another set of experiments that directly addressed the processivity of hFPGS were substrate trapping and pulse-chase experiments. These experiments utilized a pulse of [<super>14</super>C]-lometrexol(TM) and a chase or trap of unlabeled lometrexol(TM) and provided a direct measure of folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS catalyzes the processive addition of approximately four glutamate residues onto the antifolate lometrexol(TM).