Restriction Enzymes 117oligodeoxynucleotide substrates have much higher K m values
(78, 83, 84, 8 7, 92, 93), presumably because they do not fill the complete
DNA binding site of the enzyme, do not adopt the same conformation
as when part of a high-mol-wt DNA molecule, and because of the
absence of facilitated diffusion processes. Pohl et al. (94) have noticed
that there is a correlation between K m values and the length of the
recognition sequence: Restriction enzymes recognizing a hexanucleo-
tide sequence have on average a 10 times lower K m than enzymes that
recognize a tetranucleotide sequence.2.3.2. -Rate of_Reaction
Restriction enzymes are remarkably slow enzymes, with turnover
numbers (l%at) around 1-10 min -1. It has been argued that this is because
of the physical constraint that kcat/K m cannot exceed the diffusion
controlled limit for a bimolecular association rate constant (14,95),
which means that with a K m of 1 nM kca t cannot be significantly larger
than 1 s -1. As a matter of fact, single turnover experiments with EcoRI
have shown that the intrinsic rate constants of phosphodiester bond
cleavage (k' and k" in Fig. 1) come close to this limit (83,96-98). The
disparity between kca t and k' or k" as measured in steady-state and
presteady-state experiments with EcoRI and pBR322 indicates that it
is not the phosphodiester bond cleavage that is rate limiting for the
enzymatic turnover. Depending on experimental conditions, site localiza-
tion, a conformational change of the enzyme, or product release may be
the rate-determining step for the EcoRI catalyzed cleavage of DNA.
The temperature dependence of the activity of various restriction
enzymes has been analyzed in terms of the apparent activation energy,
Ea, for the enzymatic reaction and the apparent inactivation enthalpy,
E i (94). An average value of 50 + 23 kJ/mol for E a and 190 + 75 kJ/mol
for E i was determined. Most restriction enzymes show a temperature
optimum for DNA cleavage between 25 and 45°C.
The rate of cleavage has been observed to be largely independent of
the superhelicity of the substrate (99), but to depend very much on the
sequences adjacent to the recognition site. For example, the 5 EcoRI
sites in ~, DNA are cleaved with considerably different rates (100-
102). Similar observations were made with EcoRI sites in other DNA
substrates (103,104), as well as for other restriction enzymes, e.g., PstI
(105). Studies with synthetic oligodeoxynucleotides that differ in their