Chemistry Reference
In-Depth Information
15.3.3 Movement of the Two Metal Ions During Transition State Formation
In all polymerases and nucleases reported to date, the two metal ions are jointly
coordinated by a conserved Asp and the scissile phosphate (Figures 15.3 and
15.4), and their general positions conform to the 1993 proposal
84
(Figure 15.5 ). The
precise position and separation distance between the two metal ions, however, may
vary at each reaction step according to the changes in the coordination environment.
Tuning of the two metal ions is evident in both DNA pol b and RNase H. In the
crystal structure of pol b-substrate complexes, the two metal ions are 4.0 Å apart,
and in the structure of pol b - transition state - like complexes, they become 3.4 Å
apart.
40
Similarly, the separation of the two Mg
2+
ions in the RNase H-substrate
complexes vary from 4.0Å to 4.5Å, depending on which one of the catalytically
essential carboxylates was replaced by carboxylamide.
63
When the separation is
4.5 Å, the mutant protein (D132N) is completely inactive. But the mutation (D192),
in which the metal ions are 4.0Å apart, has detectable nuclease activity in the
presence of Mn
2+
.
63
The 4.0 Å separation allows the two metal ions to fi t snugly on each side of the
scissile phosphate, as depicted by Steitz and Steitz
84
(Figure 15.5). However, the
potential nucleophile coordinated by metal ion A is 3.5Å or further away from
the target phosphorus atom in the nuclease-substrate complexes (Figure 15.5). For
nucleophilic attack to occur, the hydroxide ion probably needs to be within 2.5 Å
of the phosphorus. The nucleophile is unlikely to dissociate from metal ion A and
move over 1 Å by itself, owing to the high energy cost of removing an inner-shell
metal-ion ligand and the repulsion between the negatively charged nucleophile and
the scissile phosphate. We hypothesize that the 4.0 Å separation of the two metal
ions represents a 'resting' state, and that metal ion A moves towards metal ion B
and brings the nucleophile within striking distance for phosphoryl bond formation.
Bringing the two metal ions closer could also better neutralize the developing nega-
tive charge on the pentacovalent intermediate.
A 3.5 Å separation between two Mg
2+
ions has been observed with DNA pol b
and RNase H, when complexed with transition state mimics.
86
Interestingly, such
close juxtaposing is not observed when an Asp coordinating both metal ions is
replaced by Asn (D132N).
86
In addition, two Mg
2+
ions are reported to be as close
as 3Å in the T7 RNA polymerase-substrate complex structure determined at
2.88 Å
49
(Figure 15.3A). It is possible to bring two Mg
2+
ions closer than 4.0Å
because its interatomic distance is 3.197 Å.
87
Ca
2+
has been widely used to substitute
for Mg
2+
during enzyme-substrate complex formation because it usually supports
substrate binding. Catalysis of all polymerases and the nucleases mentioned in this
chapter is inhibited by Ca
2+
.
67,88 - 90
Comparisons of crystal structures of nucleases
and polymerases reveal little difference between Ca
2+
and Mg
2+
coordination
geometries.
67,91,92
Interestingly, the interatomic distance of Ca is 3.94Å,
87
and the
closest approach between two Ca
2+
ions observed to date is 3.8 Å.
93,94
This could be
one reason that Ca
2+
is unable to support catalysis of two-metal-ion-dependent
phosphoryl transfer reactions. In contrast, the size and charge density of Zn
2+
, Co
2+
,
Cd
2+
and Mn
2+
are comparable to Mg
2+
, and these divalent cations can substitute for
Mg
2+
in catalysis.
