Recent experimental observations support the assumption that all families of polynucleotide polymerases have a universal "two-metal-ion" mechanism of nucleotide addition. This mechanism provides a general picture of the nucleotidyl transfer reaction. However, the detailed reaction pathway is still a matter of debate. We investigated two potential reaction pathways for DNA polymerase β using density-functional theory. Our model consists of 67 atoms of the polymerase active site and includes all major features thought to be important for catalysis. The first mechanism we investigated involves the formation of a PO 3 intermediate. This intermediate is thought to be involved in phosphate reactions in solution and could be accommodated in the polymerase β active site. However, the barrier to formation of this intermediate is 37.0 kcal/mol, and we do not expect that this mechanism is the one that occurs in the enzyme. The second mechanism that leads to a pentacoordinated intermediate appears to be feasible. This stepwise mechanism has relatively low barriers and, after the nucleophilic attack, every step of the reaction is exothermic. The rate-limiting step of the reaction is the nucleophilic attack, which needs 13 kcal/mol of activation energy. We predict that the barrier of the corresponding transition state, which is ionic, can be further lowered by taking into account electrostatic stabilization coming from the rest of the protein.
KNI-272 is a peptidomimetic transition state analog inhibitor,
having very high specificity and binding
affinity for the HIV-1 protease. In order to understand the
interactions that enhance drug binding to the protease,
we recorded 2D water/NOESY and water/ROESY spectra to identify water
molecules that bind tightly to the protease/KNI-272 complex. Well-ordered water molecules are observed at the
protease/inhibitor interface in the crystal structure
of the complex that have short interproton distances to the Ile50/150,
Ala28/128, and Asp29/129 amide protons.
The cross peaks between these protein protons and water protons,
observed in water/NOESY and water/ROESY
spectra, provide strong evidence that these water molecules are present
in the solution structure of the complex.
Analysis of measured NOE and ROE cross relaxation rates indicates
that, in solution, these water molecules have
long residence times, at least 1 ns and possibly greater than 7 ns.
The presence of long-lived hydration water molecules
at the protein/inhibitor interface suggests that interactions involving
these water molecules contribute to the potency
of the inhibitor. Hence, consideration of the potential role of
hydration water molecules in stabilizing protein/inhibitor structures could contribute to improved drug design and to a
better understanding of the mechanisms of
drug resistance.
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