Alteration of the properties of chicken liver dihydrofolate reductase as a result of modification by tetrathionate.

When chicken liver dihydrofolate reductase reacts with an excess of sodium tetrathionate, 1 mol of thiosulfate becomes covalently attached to the single sulfhydryl group of the enzyme. The identity of the thiosulfate was established by obtaining 1:l binding independently with both innerand outer-labeled [36S]tetrathionate. The thiosulfate form of the enzyme has 5to 10-fold more activity than native enzyme when measured with its normal substrate and cofactor, dihydrofolate and TPNH. With folate and TPNH, a slight increase (approximately 2-fold) is seen while, with dihydrofolate and DPNH, a 20% decline in activity occurs. Modification also changes the K, values, with increases for TPNH and dihydrofolate and decreases for folate and DPNH, when measured near their respective pH optima. Although the thiosulfate enzyme is stable at 4°C for 1 week or at -80°C for at least several months, it is very sensitive to heat. Both dihydrofolate and TPNH afford some protection, dihydrofolate being by far the most effective. The thiosulfate group can be removed by treatment with [36S]mercaptoethanol to yield a [36S]mercaptoethanol adduct which has approximately 35% less activity than the original free sulfhydryl form of the enzyme. Both dihydrofolate and TPNH can completely block this reaction if present in excess over enzyme. The thiosulfate group can also be replaced by its analog thiophosphate. This form is only 1.5 times more active than the unmodified enzyme (using dihydrofolate and TPNH as substrates) and, in contrast to the thiosulfate enzyme, returns to the free sulfhydryl form upon treatment with mercaptoethanol.