Direct photoaffinity-labelling of human deoxycytidine kinase with the feedback inhibitor dCTP.
Deoxycytidine kinase (dCyd kinase, EC 2.7.1.74) is a key enzyme in the salvage pathway of deoxyribonucleosides, and the human enzyme is a dimer of two 30 kDa polypeptides with a broad substrate specificity, phosphorylating both purine and pyrimidine nucleosides and using various nucleoside triphosphates as phosphate donors. The enzyme is efficiently feedback-inhibited by dCTP, which presumably is the main regulator of its activity in vivo. Submicromolar concentrations of [32P]dCTP could be used for direct photoaffinity labelling of pure dCyd kinase isolated from leukaemic spleen. A clearcut saturation of photoincorporation occurred with half-maximal incorporation at 0.07 microM-dCTP. However, the total molar incorporation of dCTP was very low (approx. 0.1%), in part due to a substantial u.v. inactivation of the enzyme. Proteinase digestion of labelled enzyme showed that dCTP was incorporated predominantly into a single peptide. Addition of equimolar concentrations of dCyd or dCMP as compared with dCTP inhibited photoincorporation approx. 50%. The presence of other nucleoside substrates, as well as phosphate donors, also inhibited photolabelling of the enzyme. Thus photoincorporation of dCTP seems to occur at a site which can bind both the phosphate donors and acceptors of dCyd kinase, which strongly support the hypothesis that dCTP functions as a multi-substrate analogue, binding and bridging both substrate sites of the enzyme.
Jansson
O
O
Medical Nobel Institute, Department of Biochemistry I, Karolinska Institute, Stockholm, Sweden.
Eriksson
S
S
D000345:Affinity Labels; D003842:Deoxycytidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D005246:Feedback; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D009705:Nucleosides / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism*; D010777:Photochemistry; D013154:Spleen / Q000201:enzymology; D013942:Thymine Nucleotides / Q000378:metabolism
D000345:Affinity Labels; D003845:Deoxycytosine Nucleotides; D046911:Macromolecular Substances; D009705:Nucleosides; D009711:Nucleotides; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D010770:Phosphotransferases; D003842:Deoxycytidine Kinase; C024157:thymidine 5'-triphosphate