Kinetic studies on the reaction catalyzed by DNA ligase from calf thymus

Kinetic analysis of the reaction catalyzed by calf thymus DNA ligase (EC 6.5.1.1) has been carried out using [5'-32P]nicked DNA as substrate. The results of initial velocity and product inhibition studies indicate that the ligase reaction is likely to proceed through the 'uni-uni uni-bi ping-pong' mechanism. The order of substrate addition and product release is as follows: ATP, PPi, nicked DNA, sealed DNA and 5'-AMP. The true Km values for ATP and for nicked DNA (5'-phosphoryl ends) were 2 microM and 0.11 microM, respectively. The turnover number was estimated to be 7 sealing events per min. dATP was an inhibitor competitive with ATP (Ki = 25 microM). The addition of 0.5 mM spermine or 5 mM spermidine resulted in an increase in the apparent Km for nicked DNA as well as in the apparent V, whereas 0.1 M KCl increased only the apparent Km for nicked DNA. Neither polyamine nor KCl affected the apparent Km for ATP. The ligase reaction was not significantly affected by aphidicolin and various phosphate compounds tested.