Chemistry Reference
In-Depth Information
Figure 15.6
(Plate 17)
Coordination of the two metal ions changes during RNA cleavage by
RNase H: (A) Enzyme-substrate complex; (B) Enzyme-product complex (See colour plate
section)
ion B in the substrate complex to a favourable six-ligand coordination in the product
complex indicates that the two metal ions also play a role in product formation. The
two metal ions jointly coordinated by the scissile phosphate may also facilitate
product release. After splitting into 5
- OH, the cleaved scissile
phosphate can no longer coordinate the two metal ions simultaneously. At least one
metal ion has to be displaced or released, as observed in product complexes of DNA
and RNA polymerases and nucleases.
49,50,73,86,91
The RNase H-product complexes
have been trapped in the crystal form by a key residue for product release (E188)
being substituted by Ala. In the product complex the cleaved scissile phosphate
moves out of the active site, and concomitantly metal ion A moves by 2 Å and retains
only one ligand from RNase H.
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Presumably eventual dissociation of the metal ion
leads to the release of the cleavage product.
Instead of having separate responsibilities, as initially proposed, the two metal
ions appear to work together throughout phosphoryl transfer reactions. It is widely
accepted that enzymes facilitate chemical reactions by proper alignment of sub-
strates relative to each other and to the catalytic residues, thereby lowering the
energy barrier between the substrate and product states. As such, the key element
of two-metal-ion catalysis may be the proper alignment of the two metal ions with
regard to the conserved carboxylates and substrate. The strong conservation of two-
metal-ion coordination in polymerases, nucleases and group I intron (in which a
backbone phosphate replaces the conserved Asp to coordinate both metal ions)
101
certainly supports this metal-ion centric view. Once the proper alignment is formed
and the enzyme-substrate complex is destabilized, a nucleophile can form and be
activated in various ways without a conventional general base. Similarly, after the
reaction proceeds past the pentacovalent intermediate, protonation of a leaving
group is energetically favourable, even without a general acid. This view is supported
by the biochemical and computational studies of metal-ion-dependent proton trans-
fer in DNA and RNA polymerases.
102 - 104
′
- phosphate and 3
′
