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but they are also correlated to positioning of the scissile phosphate. Only when the
scissile phosphate is appropriately bound in the active site are two divalent cations
jointly coordinated by the phosphate and carboxylates for catalysis, as shown in
Figure 15.4 A.
For type IIP REases, both the conformation of the active site residues and the
binding of divalent cations depend on the DNA substrate. A cognate DNA sequence
is required for two-metal-ion binding in the canonical positions, and, in return,
divalent cations are required for high-affi nity binding of the DNA substrate.
78 - 80
Without divalent cations, EcoRV binds DNA with low sequence specifi city, and in
the presence Ca
2+
, which mimics Mg
2+
for substrate binding, but inhibits hydrolysis,
EcoRV binds the cognate recognition sequence specifi cally.
81
Structural investiga-
tion revealed that when BamHI is associated with the sequence GAATCC (one
base pair substitution of the cognate GGATCC sequence), although the binding is
tight enough for crystallization, the DNA is shifted away from the binding site of
the cognate sequence and metal ions are absent.
78
Moreover, the conserved Asp,
65
which coordinates both metal ions in MutH, BamHI and BglI, is located on a loop
that is disordered in the absence of a cognate DNA, partially ordered in the pres-
ence of DNA, and fully ordered only when both metal ions are bound.
67,78
Without
divalent cations, the catalytic residues of EcoRI in all DNA-complex structures,
although ordered, are oriented in such a way that they are incapable of catalysis.
72
MutH cleaves the unmethylated strand in the hemimethylated GATC sequence
10-20-fold better than the unmethylated duplex. In the crystal structures of the
MutH-DNA complexes, the active site is clearly better ordered and better oriented
for catalysis with the hemimethylated GATC than the unmethylated.
67
Both the
active site conformation and the metal ion binding in MutH-like nucleases are
consistently shown to depend on binding of a cognate DNA substrate.
The dependence of the nucleases on the cognate DNA substrate for metal-ion
binding provides an explanation as to why the number and location of metal ions
in the structures of homologous type IIP REases vary. To crystallize nuclease-
substrate complexes, cleavage has to be prevented by artifi cial means, for example,
substitution of catalytic residues, use of inert substrate analogues, or divalent cations
that do not support catalysis. Strategies used to capture enzyme-substrate com-
plexes with the purpose of preventing chemistry from taking place inevitably perturb
the protein-DNA interactions. In addition, crystal lattice contacts may distort mac-
romolecular interactions. One has to interpret crystal structures and particularly the
active site conformation, including the number and location of metal ions, with
caution. The exquisite sensitivity of metal ions to alterations in coordination envi-
ronment probably underlies the specifi city of these enzymes.
15.3.2 Two Metal Ions Enhance Catalytic Specifi city
When two divalent cations were fi rst observed in the active site of alkaline phos-
phatase and the exonuclease of the Klenow fragment,
82,83
they appeared to mediate
binding of nucleic acids in the carboxylate-rich active site. The two metal ions are
located roughly in parallel to the phosphosugar backbone on the opposite side of
