Abstract

Folate reductase of amethopterin resistant S-180 cells (AT/3000), grown in vitro, is inactivated by o-phenanthroline, EDTA, p-chloromercuribenzoate, iodoacetamide, trypsin, subtilisin, elastase, and carboxypeptidase B, but not by carboxypeptidase-A or leucineaminopeptidase. The enzymatic activity and the number of amethopterin binding sites were always lost in parallel fashion. When substrates or coenzymes were present, complete protection of the enzymic activity and of the amethopterin binding sites against all these agents was observed. Competitive inhibitors, 2,4-diaminopurine and -pyrimidine, which mimic just a small portion of the substrate molecule, also protected completely against o-phenanthroline and against the proteolytic enzymes but poorly against SH-reagents. It is suggested that folate (probably the pyrimidine portion of it) and TPNH are bound to the enzyme in the proximity of a metal and SH-groups. The protection against the proteolytic digestions is probably provided through conformational changes in the enzyme. No major proteolytic breakdown of the enzyme molecule, as a whole, was observed when folate reductase was complexed with amethopterin.

It is suggested that the intracellular presence of substrate, coenzyme or competitive inhibitor of folate reductase may alter the turnover of this enzyme by preventing the proteolytic breakdown. In certain circumstances this could lead to an increase in the folate reductase content of cells and tissues. Also, it is concluded that folate reductase can form binary complexes with both the substrate and the coenzyme. Thus, the prior presence of coenzyme on the enzyme surface is not necessary for the attachment of the substrate and vice versa. Since the concentrations of folate, TPNH, and TPN which afforded 50% protection were of the same order of magnitude as the respective K_m values, it seems that the presence of one on the enzyme surface does not affect the affinity to the other.