Chemistry Reference
In-Depth Information
coordination and the preference of substrates may explain why polymerases exhibit
strong substrate specifi city that surpasses selection by base pairing.
The rate-limiting step in polymerization is postulated to be the key step for
rejecting a wrong incoming nucleotide and ensuring replication fi delity.
53
A confor-
mational step prior to the chemical reaction was previously suggested to be the
rate-limiting step in DNA synthesis.
54
Because of the structural observation of a
large fi nger-domain movement induced by binding of a correct incoming nucleotide
in Taq and T7 DNA pol and in HIV RT that transforms the active site from an
' open ' to the ' closed ' state,
41,45,55
the fi delity of DNA polymerases was originally
attributed to such an induced-fi t conformational change. In recent years, this hypoth-
esis has been challenged by presteady-state kinetic analyses of A, B and X family
DNA polymerases.
53
It has been shown that the fi nger domain movement or closing
of the active site is much faster than the phosphoryl transfer reaction and therefore
is not the rate-limiting step.
56 - 60
Furthermore, analogous domain movement is not
observed in all polymerases, for example, the X family pol l and all Y family
members, which still exhibit respectable fi delity.
27
These observations lead to the
verdict that a step after dNTP binding and the fi nger - domain movement is the
rate - limiting step.
A rate-limiting conformational change that differentiates the correct versus
incorrect incoming nucleotide has been observed for all polymerases, including
Dpo4,
61
whose active site doesn't undergo fi nger - domain movement. The conforma-
tional step therefore has to be other than the domain movement and is likely subtle.
The displacement of metal ions observed in the Dpo4-substrate complexes when
the 3
-OH nucleophile is absent or the replicating base pair is a mismatched, sug-
gests that binding of two Mg
2+
ions in the active site is insuffi cient for nucleotide
incorporation. The proper positioning and alignment of the two metal ions with
regard to the catalytic residues and substrate may be the rate-limiting conforma-
tional changes and the key to converting small differences in binding energy to large
differences in catalytic effi ciency in all polymerases.
′
15.3 Nucleases That Require Two Mg
2+
Ions in the Active Site
RNase H and MutH represent two large superfamilies of nucleic acid enzymes that
break backbone phosphodiester bonds using two-Mg
2+
ion catalysis. The RNase H-
like superfamily includes the transposases and retroviral integrases with a DDE
motif, argonaute endonuclease and the Holliday junction resolvase RuvC.
12,62
The
catalytic domains feature a central mixed b - sheet surrounded by a - helices (Figure
15.4A). Like DNA and RNA polymerases, two absolutely conserved Asps are the
hallmark of the RNase H-like nucleases and transposases.
63,64
They are located on
two adjacent parallel b-strands, one long and one short (Figure 15.4A). One or two
additional carboxylate residues (Asp or Glu) located on the surrounding secondary
structures may be required for the catalysis. The MutH-like superfamily includes
the type IIP restriction endonucleases (REases, see the comprehensive review
65
)
and phage lambda Exo I.
66
The catalytic centre marked by the DEK motif is located
