Restriction Enzymes 115
accelerates the rate of the cleavage reaction (64-67) by lowering the
apparent K m. Linear diffusion along the DNA may also be responsible
for the processive cleavage of DNA in two (or more) sites by a restric-
tion enzyme: Depending on reaction conditions, in particular the ionic
strength, two sites close to each other may be cleaved in a processive
manner, when the enzyme remains associated with the DNA after the
first cleavage, rather than in a distributive manner, when the enzyme
dissociates after the first cleavage (68-70).
2.3.1. Affinity
Although Mg 2+ ions are necessary for the phosphodiester bond cleav-
age, they are not needed for the strong binding of restriction enzymes
to DNA (see note added in proof at end of chapter). The affinity of
restriction enzymes for their substrate in the absence of DNA is very
high: measurements carried out with macromolecular DNA indicate
that, e.g., EcoRI binds to its site with an equilibrium constant of the
order of 101°-1011M -I, to nonspecific DNA with an equilibrium con-
stant of the order of 106M -1 (71, 72). Similar results have been obtained
with dodekadeoxynucleotides or larger oligodeoxynucleotides (72, 73),
but not, however, with smaller ones, which are not bound as firmly
(74). Obviously, oligodeoxynucleotides that are too small to fill the
complete DNA binding site of the restriction enzyme are not bound
with the maximum affinity. It is noteworthy that, for all restriction
enzymes analyzed in this context, residues external to the recognition
site are important for enzyme-DNA interaction. The strength of bind-
ing between EcoRI and DNA decreases with increasing ionic strength
(60, 72, 73) demonstrating that electrostatic interactions are involved
in complex formation. Although so far no precise data have been obtained
for the affinity of restriction enzymes for their substrate in the pres-
ence of Mg 2÷ ions, presteady-state (75) as well as steady-state experi-
ments indicate that restriction enzymes display a high affinity for their
substrate. Apparent K,,, values determined for eight restriction enzymes
and high-mol-wt substrates range from 0.5-17 nM (Table 3). Although
these values have been determined with different substrates, i.e., sub-
strates with variable proportions of nonspecific and specific DNA
sequences, as well as under considerably different buffer conditions
and temperatures, they demonstrate that restriction enzymes in gen-
eral have a very low K m for high-mol-wt DNA. In contrast,