Open AccessKinetics and Effect of Salts and Polyamines on T4 Polynucleotide Ligase
Author(s) -
RAAE Arnt J.,
KLEPPE Ruth K.,
KLEPPE Kjell
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb21021.x
Subject(s) - spermine , substrate (aquarium) , dna ligase , polynucleotide , spermidine , chemistry , kinetics , dna , enzyme , biochemistry , enzyme kinetics , stereochemistry , active site , biology , ecology , physics , quantum mechanics
The kinetics of T4 polynucleotide ligase has been investigated at pH 8, 20 °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 K m for the DNA substrate was 0.6 μM and that of ATP 100 μM. 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 70 mM, of salts such as KC1, NaCl, NH 4 Cl and CsC1. 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 K m for the DNA substrate whereas the apparent K m 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.