Kinetics and effect of salts and polyamines on T4 polynucleotide ligase.
The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - [(DT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002103:Cadaverine / Q000494:pharmacology*; D003090:Coliphages / Q000201:enzymology*; D003959:Diamines / Q000494:pharmacology*; D011756:Diphosphates / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D009994:Osmolar Concentration; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011700:Putrescine / Q000494:pharmacology*; D012492:Salts; D013095:Spermidine / Q000494:pharmacology*; D013096:Spermine / Q000494:pharmacology*
D003959:Diamines; D011756:Diphosphates; D012492:Salts; D013096:Spermine; D000249:Adenosine Monophosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D011118:Polynucleotide Ligases; D002103:Cadaverine; D013095:Spermidine; D011700:Putrescine