60 A. Gabibov et al.
simply not relevant for the description of the kinetics. In contrast, the in-
teraction of relaxed DNA and topoisomerase I obeys the Michaelis–Menten
kinetics.
As a result,relaxingscDNA exists mainly in the form of the covalent com-
plex with an enzyme, and therelaxed one – in a non-covalent enzyme-DNA
complex. In this connection the addition of the inhibitors, e.g. campthotecine,
during a process of topoisomerisation would produce significantly more irre-
versible nicks in the DNA chains than the addition of the same reagent at
a point when the process is over. Similar explanation can also be given to
earlier experiments by stopping the topoisomerisation reaction by detergents,
to the studies of the processivity of this reaction, as well as to a competitive
inhibition with DNA of different topology, etc.
The fact that the topoisomerisation reaction is described by the first-
order kinetics must mean that the reaction is limited by some monomolec-
ular reaction whose rate constant does not change through the whole course
of the experiment (and, hence, is the same for either of the topoisomers). In
low processivity conditions (high salt,μ> 0 .2), most likely, the rate of the
phosphodiester bond cleavage is a limiting step for topoisomerisation. In this
connectionk′=kc 1 E/S, and hence, ifE=S, thenk′=kc 1. We performed
k′measurements in a series of experiments with increasing DNA-enzyme ratio
(Fig. 4.10). The extrapolation of the dependency obtained towards to a value
ofE/S= 1 gave a value ofkc 1 =0.08, which in turn, was close to a value of
the similar constant calculated for the vaccine topoisomerase using oligonu-
cleotide as a model substrate [26].
The dissociation of the enzyme-product complex could also be a rate-
limiting step (e.g. low salt conditions). However, this process plays a sub-
stantial role only in the case when a large portion of DNA has been already
Fig. 4.10.Dependence of the effective constant of pTM relaxation catalysed by
topoisomerase I on different enzyme/DNA ratio at 100μm NaCl