Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues.
Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.
Cieśla
J
J
Nencki Institute of Experimental Biology, Warsaw, Poland.
Gołos
B
B
Dzik
J M
JM
Pawełczak
K
K
Kempny
M
M
Makowski
M
M
Bretner
M
M
Kulikowski
T
T
Machnicka
B
B
Rzeszotarska
B
B
D000818:Animals; D005468:Fluorodeoxyuridylate / Q000031:analogs & derivatives / Q000494:pharmacology; D006926:Hymenolepis / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000469:parasitology; D008115:Liver Regeneration; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013763:Tetrahydrofolates / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*
D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase