Methods in Molecular Biology • 16 Enzymes of Molecular Biology

114 Pingoud, Alves, and Geiger

linear

diffusion

(~ I I .~-

",,. (~ (k')

specific non-1~ / specific 1~

binding binding

cleavage cleavage

1st strand 2nd strand

nicked inter- product

mediate release

II il I~ i~

+ + + +

0 0 0 0

Fig. 1. Generalized scheme to illustrate the events following the association of a
macromolecular DNA with a restriction enzyme.

Figure 1 depicts the reaction catalyzed by a restriction enzyme. It
shows (exemplified for EcoRI) that this enzyme interacts not only
with its recognition sequence, but also with lower affinity with non-
specific sequences (60,61). This results in competitive inhibition of
the enzyme. Assuming that Michaelis-Menten conditions are valid,
the steady-state rate of reaction, v, is given by:

v = [Vma x • F • Ct°tal]/[g m • F + C t°tal] F = 1/[1 + L • Km/Ki] (3)

where V,,~x and K m are the intrinsic maximum velocity and Michaelis
constant, K i, is the inhibitory constant of the nonspecific DNA, L is the
relative concentration of nonspecific over specific DNA and C t°tal the
sum of the total concentration of specific and nonspecific DNA (62).
This implies that the addition of DNA not containing a recognition
sequence (as a matter of fact, single-stranded DNA and RNA have a
similar effect !) to a DNA substrate leads in a concentration-dependent
manner to an inhibition of the cleavage reaction (62,63). On the other
hand, nonspecific binding might under certain conditions help restric-
tion enzymes to localize their specific recognition site by facilitated
diffusion, provided nonspecific and specific sites are on the same
DNA molecule. It has indeed been demonstrated that nonspecifically
bound restriction enzymes slide along the DNA in an apparently ran-
dom process until the specific sequence is reached, a process that