Tim Fitzwater via methods%40net.bio.net (by tfitzwater from somalogic.com)
Mon Jun 30 09:49:53 EST 2008

Lillehaug, J. R. and K. Kleppe (1975). "Kinetics and specificity of T4
polynucleotide kinase." Biochemistry 14(6): 1221-5.

                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


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