INHIBITION OF FOLYLPOLYGLUTAMATE SYNTHETASE BY SUBSTRATE-ANALOGS WITHAN ORNITHINE SIDE-CHAIN

4-[[(4-Aminopteridin-6-yl)methyl]amino]benzoyl]-L- ormithine (dAPA-Orn ) was synthesized, and its ability to inhibit folylpolyglutamate synth etase from mouse liver was compared with that of the corresponding 2,4 -diamino analogue APA-Orn. Also compared were the inhibitory activitie s of the deaza analogues 5-deazaAPA-Orn, 8-deazaAPA-Orn, and 5,8-didea zaAPA-Orn, as well as those of N-alpha-pteroyl-L-ornithine (PteOrn) an d its deaza analogues 5-deazaPteOrn and 5,8-dideazaPteOrn. The inhibit ion constant K-i of dAPA-Orn was 7-fold greater than that of APA-Orn, indicating that the 2-amino group plays a role in binding to the activ e site. The binding affinity of the 2,4-diamino compounds increased in the order 5-deazaAPA < APA-Orn < 5,8-dideazaAPA-Orn < 8-deazaAPA-Orn, and that of the 2-amino-4(3H)-oxo compounds increased in the order 5- deazaPteOrn < PteOrn < 5,8-dideazaPteOrn. The most potent inhibitor of both groups was 8-deazaAPA-Orn, with a K-i of 0.018 mu M, correspondi ng to an 8-fold and 15-fold increase in affinity relative to APA-Orn a nd 5-deazaAPA-Orn, respectively. The results suggest (a) that the bind ing of Orn-containing folylpolyglutamate synthetase inhibitors is affe cted to a greater degree by replacement of N-8 by a carbon atom than i t is by the corresponding change at N-5, (b) that the effect of carbon for nitrogen replacement is greater in the 2,4-diamino derivatives th an in the 2-amino-4(3H)-oxo compounds, and (c) that the 2,4-diamines a re the better inhibitors. Comparison of the K-i values of the Orn-cont aining inhibitors with the K-m values of the corresponding glutamate-c ontaining substrates revealed that K-m/K-i ratio can vary as much as 1 00-fold depending on the nature of the heterocyclic moiety, suggesting that caution should be exercised in using K-m values of known substra tes to predict K-i values of putative inhibitors.