Purification and Properties of Deoxythymidine Kinase Induced by Bacteriophage T4 Infection1

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Abstract
Deoxythymidine kinase [EC 2.7.1.2 1] activity in Escherichia coli increased in response to infection with T even phages but not with T odd phages. This was due to the formation of a new enzyme distinct from the deoxythymidine kinase of E. coli. Among the phages tested, T4 am N82 (gene 44) induced the enzyme most strikingly. Phage-induced deoxythymidine kinase was purified 440-fold from a crude extract of T4 am N82-infected E. coli KY896 (tdk−: deoxythymidine kinase deficient) by streptomycin treatment, ammonium sulfate fractionation, and DEAE- and phosphocellulose chromatographies. The molecular weight of the enzyme was estimated to be about 86,000 by Sephadex G-200 gel filtration. The enzyme seemed to be composed of subunits with a molecular weight of about 28,000. The partially purified enzyme had a pH optimum at 6.8 and was stimulated several-fold by Mg2+, Mn2+, and Co2+. Deoxythymidine served as a good phosphate acceptor but other deoxynucleosides and 5-halogenated deoxyuridines did not. As phosphate donors, dATP and dGTP were about 50% as active as rATP. dTTP was a potent inhibitor of the enzyme, and the inhibition was highly dependent on pH. No remarkable activation was found with various nucleotides. The saturation curve for ATP was sigmoidal. Only one Km value for deoxythymidine (7.0×10−5 M) was obtained from Lineweaver-Burk plots with an ATP concentration of 1.43 mM, whereas two Km values (3.3×10−5 M and 2.4×10−4 M) were found with 0.46 mM ATP. When dTTP was present, two Km values (7.0×10−5M and 4.2×10−4M) were found even at 1.43 mM ATP. Unlike the E. coli enzyme, T4 deoxythymidine kinase was inactivated quickly above 50°C but was stable at moderate temperatures, and no drastic conformational change was detected even though in the presence of regulatory nucleotides.