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A binding method that detects only the strongest binding site for a ligand on a protein has been used to show that folates and folate analogs, conjugated with poly-gamma-glutamates, are bound to hemoglobin. When the concentration of hemoglobin is much larger than that of the polyglutamate, as is the case in the red cell, the fraction bound is a direct function of the hemoglobin concentration and is independent of the total polyglutamate concentration. Binding to deoxyhemoglobin tetramers is competitive with 2,3-diphosphoglycerate. In oxyhemoglobin the folyl and methotrexate polyglutamates are bound preferentially by free alpha beta dimers, but removal of the pteridine moiety leads to tetramer binding even in oxyhemoglobin. Changes in the length of the polyglutamate side chain and alterations of the pteridine structure such as reduction and/or methylation have a much larger effect on the constant for binding to deoxyhemoglobin tetramers than on that for oxyhemoglobin dimers. The implications of these results for the storage of pteroylpolyglutamates in the erythrocyte and their release from the red cell under the influence of the degree of oxygenation and variations in the 2,3-diphosphoglycerate level are discussed.

The ability of folate analogues to inhibit host and viral thymidylate synthetases was measured using the corresponding Escherichia coli and T2-phage-induced enzymes. In the absence of Mg2+, 6 x 10(-7) M pteroylhexaglutamate inhibited the T2-phage-induced synthetase by 50%, but at least 100-fold greater levels of this compound were necessary to inhibit the E. coli synthetase by this amount. At 2.5 x 10(-6) M pteroylhexaglutamate, at least 80% inhibition of the T2-phage synthetase could be obtained with little or no inhibition of the E. coli enzyme. The pteroylmonoglutamate was about 2 orders of magnitude less inhibitory towards the T2-phage enzyme than the pteroyltri- to -heptaglutamates. However, upon addition of Mg2+ to the assay mixture, the inhibition produced by pteroylhexaglutamate was essentially reversed, with the E. coli synthetase now increasingly inhibited by this compound and the T2-synthetase only minimally impaired. Methotrexate and N10-formyl-2-amino-4-hydroxyquinazoline, although inhibitory to both enzymes in the presence or absence of Mg2+, did not show this differential selectivity. These results suggest that certain folate analogues may be useful in distinguishing between a host and an infecting organism's thymidylate synthetase and could thus provide an additional means of screening for potential chemotherapeutic agents.

Formiminotransferase-cyclodeaminase, a circular tetramer of dimers, binds four tetrahydropteroylpolyglutamates/octamer, which indicates that these polyglutamate sites are formed by one type of subunit interface. The transferase and deaminase are separate catalytic sites as determined by inhibition studies with (6R)-tetrahydropteroylglutamate and by the observation that the activities can operate simultaneously. Under conditions where the transferase is saturated with tetrahydropteroyl(glutamate)n substrate, exogenously added formimino intermediate is utilized by the deaminase only if at least one of the substrate/intermediate pair is a monoglutamate. These properties indicate the existence of only one polyglutamate site/pair of catalytic sites. Kinetic specificity for each activity as measured by Vm/Km increases for longer polyglutamates, but does not differentiate among 4, 5, 6, and 7 glutamates. The enzyme shows distinct preference for hexaglutamate based on Kd as well as on Km values. With all substrates, Vm of the deaminase is greater than that of the transferase, allowing for potential channeling of the intermediate between active sites. Efficiency of channeling, optimal with pentaglutamate, does not correspond with affinity for binding. This demonstrates that a steric requirement predominates over simple sequestering of intermediates on the enzyme surface as the fundamental mechanism for channeling.