THYMIDYLATE SYNTHASES FROM HYMENOLEPIS-DIMINUTA AND REGENERATING RAT-LIVER - PURIFICATION, PROPERTIES, AND INHIBITION BY SUBSTRATE AND COFACTOR ANALOGS

Comparative studies of thymidylate synthases, isolated from the tapewo rm, Hymenolepis diminuta, and regenerating liver of its host, rat, aim ed at a possibility of specific inhibition of the helminthic enzyme, a re presented. While similar in structure (dimers with monomer molecula r masses of 33.7 kDa and 34.9 kDa, respectively) and parameters descri bing interactions with substrates and products, the tapeworm and rat e nzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, co mpared with the host, enzyme was less sensitive to the competitive slo w-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equ ally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N-4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. cr-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzy mes. Both enzymes differed markedly in sensitivity to inhibition by 10 -propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu( 1-3)), with pddPteGlu(1) being stronger inhibitor of the mammalian enz yme, but pddPteGlu(3) showing opposite specificity. Sulfonamidobenzoyl glutamate analogue of pddPteGlu (pddPteSO(2)Glu) and 2-desamino-2-meth yl derivative of this analogue (CH(3)pddPteSO(2)Glu) were weaker inhib itors of both enzymes than the parent compound. Substitution of the gl utamyl residue in CH(3)pddPteSO(2)Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glyc ine, valine or phenylglycine were without a distinct effect with the h ost enzyme but weakened inhibition of the tapeworm enzyme.