Chemistry Reference
In-Depth Information
15.3.4 Metal Ions A and B Together Destabilize the Substrate and
Assist Product Formation
Metal ion A has been suggested to play an essential role in nucleophile formation
in ribozymes and enzymes that lack a conventional general base.
84
Experimental
observations accumulated over the years suggest that nucleophile formation cannot
be achieved by metal ion A alone. In the absence of an apparent general base, a
potential nucleophile is often coordinated by the nucleic acid substrate, in addition
to metal ion A. For example, in RNase H, the potential nucleophilic water molecule
interacts with metal ion A and the pro-Rp nonbridging oxygen of the phosphate 3
′
to the scissile bond,
63
and in MutH, a Lys sidechain supplies the third ligand
67
(Figure
15.4B). Neither phosphate oxygen nor Lys is a conventional general base. Interest-
ingly, although the pro-Rp oxygen coordinates the nucleophile in both RNase H
and MutH, its stereospecifi c replacement with sulfur has the opposite effect. The
Rp sulfur enhances the cleavage activity of MutH,
67
but it inhibits RNase H.
95
MutH,
whose active site contains a Lys, likely benefi ts from the extra negative charge
brought by the Rp sulfur (due to the nature of the single P-S).
96
But RNase H,
whose active site contains four carboxylates, may be inhibited by the extra negative
charge and steric hindrance due to the sulfur substitution (larger atomic radius than
oxygen). Parallel to the diverse ways of substrate-assisted nucleophile formation,
the coordinating carboxylate for metal ion A can be replaced by carboxylamide or
histidine in the RNase H family.
63,97
It appears that the net charge environment
contributed by both the catalytic residues and the substrate is the key for two-
metal - ion catalysis.
Metal ion B may promote the phosphoryl transfer reaction instead of passively
stabilizing the transition state, as originally proposed.
84
Even though the A metal
ion seemingly initiates the chemical reaction by directly coordinating to the nucle-
ophile, replacement of carboxylates that coordinate metal ion A often do not elimi-
nate nuclease activities as effectively as mutating the B metal ion ligands.
63,98
In
addition, the B site also exhibits a stronger metal ion preference than the A site. In
the 3
exonuclease of DNA polymerase I, the B site normally binds a Mg
2+
ion,
99
but it rejects Mg
2+
when the nonbridging oxygen of the scissile phosphate is
replaced by sulfur.
100
In the crystal structures of RNase H-substrate complexes, the
Mg
2+
ion at the B site is coordinated irregularly by fi ve ligands, none of which is a
water molecule (Figure 15.6A).
63
The nonideal coordination geometry and ligands
devoid of water suggest that metal ion B in the enzyme-substrate complex is in a
high-energy state and may strain the scissile phosphodiester bond. The deviation
from ideal octahedral coordination geometry may explain why Mg
2+
is preferred
over Ca
2+
for catalysis. Ca
2+
readily accepts six, seven, eight or nine ligands in various
coordination geometries, and promiscuity regarding ligand type and coordination
geometry renders Ca
2+
less likely to destabilize the enzyme-substrate complex and
promote product formation. This may be another reason that Ca
2+
does not usually
replace Mg
2+
in phosphoryl transfer reactions.
In a mutant RNase H-product complex, the B-site Mg
2+
shifts by 0.9 Å and
becomes coordinated in near octahedral geometry by six ligands, two of which are
water molecules (Figure 15.6B).
86
The change from fi ve-ligand coordination of metal
′
to 5
′
