Mammalian Deoxynucleoside Kinases: IV. DEOXYTHYMIDINE KINASE: PURIFICATION, PROPERTIES, AND KINETIC STUDIES

Abstract

Abstract Deoxythymidine kinase has been purified about 130-fold from calf thymus by fractionation with streptomycin, ammonium sulfate, and protamine and by gel filtration chromatography. The molecular weight of deoxythymidine kinase was estimated to be about 55,000 by gel filtration and sucrose density gradient centrifugation. The apparent Km values for deoxythymidine and ATP were 57 and 50 µm, respectively. Among the naturally occurring nucleosides, only deoxythymidine and deoxyuridine were active as phosphate acceptors. All common nucleoside 5'-triphosphates, except dTTP, were active as phosphate donors. Deoxynucleotide derivatives of thymine were inhibitors of deoxythymidine kinase, the potency of inhibition being in the order of dTTP g dTDP g dTMP. The inhibition produced by dTTP appeared to be competitive with respect to the phosphate acceptor, deoxythymidine. With respect to the phosphate donor, the inhibition produced by dTTP was more complex, being noncompetitive at low concentrations of ATP and competitive at high concentrations of ATP. Substrates, products, and nucleotide inhibitors partially protected the enzyme from heat inactivation. Initial velocity patterns suggested that the reaction catalyzed by deoxythymidine kinase follows a sequential mechanism.