Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase.
The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.
Datta
N S
NS
Department of Internal Medicine, University Hospital, Ann Arbor, Michigan 48109-0108.
Shewach
D S
DS
Mitchell
B S
BS
Fox
I H
IH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002460:Cell Line; D003839:Deoxyadenosines / Q000494:pharmacology; D003841:Deoxycytidine / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008274:Magnesium; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology*; D014407:Tumor Cells, Cultured
D003839:Deoxyadenosines; D003845:Deoxycytosine Nucleotides; D003841:Deoxycytidine; C024107:2'-deoxycytidine 5'-triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D003842:Deoxycytidine Kinase; D008274:Magnesium