Kinetics and specificity of T4 polynucleotide kinase.
The kinetics of T4 polynucleotide kinase has been investigated at pH 8.0 and 37 degrees. Double reciprocal plots of initial rates vs. substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to the ordered sequential mechanism shown in eq 2 in the text for phosphorylation of a DNA molecule. Based on this mechanism the rate equation for the overall reaction was deduced and the various kinetic constants estimated. Hill plots indicated little or no interaction between active sites in the enzyme. The apparent Michaelis constants and V-max were determined at a fixed ATP concentration, 66 muM, for a number of different substrates varying in chain length, base composition, and nature of the sugar, and a wide variation was found. For the nucleoside 3'-monophosphates tested both the apparent Michaelis constant and V-max values were from approximately 2 to 5 times larger than for the corresponding oligonucleotide. The following orders were obtained with regard to apparent Michaelis constants and V-max for the nucleoside 3'-monophosphates investigated: Michaelis constant, rGP greater than rUp greater than rCp greater than rAp greater than dTp; V-max, rGp greater than rCp greater than rAp greater than dTp greater than rUp. Somewhat similar results were also obtained with the deoxyoligonucleotides tested.
D001667:Binding, Competitive; D003090:Coliphages / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D004267:DNA Viruses / Q000201:enzymology; D003854:Deoxyribonucleotides / Q000378:metabolism; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D013329:Structure-Activity Relationship
D003854:Deoxyribonucleotides; D009841:Oligonucleotides; D012265:Ribonucleotides; D004247:DNA; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase