Human deoxycytidine kinase purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients

Abstract

A procedure for purifying human cytoplasmic and mitochondrial deoxycytidine kinase (NTP:deoxycytidine 5′-phosphotransferase, EC 2.7.1.74) was developed. Both purified isozymes have a similar molecular weight, activation energy and catalyze the reaction by a sequential mechanism. These two isozymes differ with respect to their substrate specificities. With cytoplasmic deoxycytidine kinase, ATP, GTP and TTP have the highest reaction velocity. Pyrimidine nucleoside triphosphates have higher affinity but lower V than purine nucleoside triphosphates. Cytidine and arabinosylcytidine can serve as substrates. With mitochondrial isozyme only ATP gives the highest reaction velocity. ATP and dATP have the same K m but different V values. Besides deoxycytidine, also deoxythymidine but not cytidine or arabinosylcytidine can serve as substrates. There are also differences between these two isozymes with respect to their sensitivity to inhibition. For cytoplasmic enzyme, Br 5dCyd and Iodo 5dCyd are not inhibitory. Both dCTP and UTP are competitive inhibitors ( K i 0.25 and 0.5 μM, respectively) with respect to ATP. For mitochondrial isozyme both Br 5dCyd and Iodo 5dCyd are inhibitory and dCTP and TTP are competitive inhibitors ( K i 2 and 10 μM, respectively) with respect to ATP.