Studies on Identifying the Binding Sites of Folate and its Derivatives in Lactobacillus Casei Thymidylate Synthase

Summary

It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A prevents the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates.

To determine the region of the synthase involved in the binding of the glutamate residues, Pte[¹⁴C]GluGlu₆ was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH₂Hi₄PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two ¹⁴C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47–61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu₇ are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.

This investigation was supported in part by Public Health Research Grants GM 26387 and GM 26645 from the National Institute of General Medical Sciences, PHS/DHHS.