Interactions of platinum complexes with the essential and nonessential sulfhydryl groups of thymidylate synthetate.
Thymidylate synthetase (methylenetetrahydrofolate:deoxyuridylate C-methyltransferase) from Lactobacillus casei was progressively inactivated when incubated at 25 degrees C, pH 6.8, in the presence of trans-Pt(NH3)2Cl2. The inhibition appeared to be irreversible, and the rate ofa ctivity loss was dependent on the inhibitor concentration. The corresponding cis isomer was incapable of inhibiting the enzyme under the same conditions. The presence of 2-mercaptoethanol protected the enzyme from inhibition, but did not reactivate enzyme preparations which had been inhibited prior to the addition of the thiol. The interactions of cis- and trans-Pt(NH3)2Cl2 with the enzyme's sulfhydryl (-SH) groups were inferred from the results of spectrophotometric titrations of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate. The results suggested that the cis isomer reacted with an average of 1.3 of the enzyme's 4-SH groups and that these were not essential for catalysis. The trans isomer reacted with a total of approximately 2.5 -SH groups, 1.2 of which are essential for catalysis. Neither the trans isomer nor a combination of both isomers was able to react with 1.2 of the 4 -SH groups. Further evidence that the Pt complexes are interacting with enzyme's -SH groups was obtained by reversibly blocking the -SH groups of thymidylate synthetase, and demonstrating the resistance of these preparations to inhibition by the trans Pt complex. Possible explanations for the preferential inhibition of thymidylate synthetase by only one of the two geometric isomers of Pt(NH3)2Cl2 are considered.
D001665:Binding Sites; D002945:Cisplatin / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008780:Methyltransferases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013438:Sulfhydryl Compounds; D013940:Thymidylate Synthase / Q000378:metabolism
D006902:Hydroxymercuribenzoates; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D008780:Methyltransferases; D013940:Thymidylate Synthase; D002945:Cisplatin