Demonstration of separate binding sites for the folate coenzymes and deoxynucleotides with inactivated Lactobacilluscasei thymidylate synthetase

It is revealed that thymidylate synthetase can be inactivated in a manner that does not prevent the binding of the substrates individually.

Abstract

In contrast to (+)5,10-methylenetetrahydropteroylmonoglutamate which does not bind to Lactobacilluscasei thymidylate synthetase, the corresponding tetraglutamate analog binds to a single site with a KD = 2 × 10−5 M. Alkylation of one of the enzyme's four cysteines with N-ethylmaleimide or iodoacetate prevented the binding of dUMP, but did not affect the binding of the pteroyltetraglutamate. Inactivation of the synthetase with carboxypeptidase A, however, prevented the binding of (+)5,10-methylenetetrahydropteroyltetraglutamate but not that of dUMP. The binding of (+)5,10-methylenetetrahydropteroyltetraglutamate to native enzyme was associated with the appearance of a positive circular dichroic band at 303 nm ([θ] = 7 × 104 deg·cm2dmol−1). The latter effect was not impaired by the inhibition of the enzyme with N-ethylmaleimide, whereas formation of the ternary complex, coenzyme-synthetase-FdUMP, was prevented by alkylation. These studies reveal that thymidylate synthetase can be inactivated in a manner that does not prevent the binding of the substrates individually.