Human carbonic anhydrases (HCAs) are responsible for
the pH control
and sensing in our body and constitute key components in the central
pH paradigm connected to cancer therapeutics. However, little or no
molecular level studies are available on the pH-dependent stability
and functional dynamics of the known isozymes of HCA. The main objective
of this Article is to report the first bench-marking study on the
structure and dynamics of the two most efficient isozymes, HCA II
and IX, at neutral pH using classical molecular dynamics (MD) and
constant pH MD (CpHMD) simulations combined with umbrella sampling,
transition path sampling, and Markov state models. Starting from the
known crystal structures of HCA II and the monomeric catalytic domain
of HCA IX (labeled as HCA IX-c), we have generated classical MD and
CpHMD trajectories (of length 1 μs each). In all cases, the
overall stability, RMSD, and secondary structure segments of the two
isozymes are found to be quite similar. Functionally important dynamics
of these two enzymes have been probed in terms of active site hydration,
coordination of the Zn(II) ion to a transient excess water, and the
formation of putative proton transfer paths. The most important difference
between the two isozymes is observed for the side-chain fluctuations
of His-64 that is expected to shuttle an excess proton out of the
active site as a part of the rate-determining intramolecular proton
transfer reaction. The relative stability of the stable inward and
outward conformations of the His-64 side-chain and the underlying
free energy surfaces are found to depend strongly on the isozyme.
In each case, a lower free energy barrier is detected between predominantly
inward conformations from predominantly outward ones when simulated
under constant pH conditions. The kinetic rate constants of interconversion
between different free energy basins are found to span 10
7
–10
8
s
–1
with faster conformational
transitions predicted at constant pH condition. The estimated rate
constants and free energies are expected to validate if the fluctuation
of the His-64 side-chain in HCA IX may have a significance similar
to that known in the multistep catalytic cycle of HCA II.
We present in this article a case study on the thermodynamics of binding to human carbonic anhydrase II (HCA II) by three well-known inhibitors, viz. (a) acetazolamide (AZM) that directly binds to the catalytic Zn(II) ion at the active site, (b) non-zinc binding 6-hydroxy-2-thioxocoumarin (FC5) (c) 2-[(S)benzylsulfinyl]benzoic acid (3G1). In each case, the crystal structure or its analogue of inhibitor-bound HCA II has been used to perform classical molecular dynamics (MD) simulation in water till 1 ms. AZM and FC5 are found to undergo repeated binding and unbinding with markedly different dynamics from the partially buried, substrate-binding hydrophobic pocket near the active site. 3G1, on the other hand, is found to remain mostly at its crystallographic binding site occluded from the active site of HCA II. The associated binding free energies ( DG bind;solv ) have been computed using the known MM/GBSA method and compared to the available experimental data. Our results show that DG bind;solv encounters several issues including limited sampling of multiple binding sites and incorrect prediction of the affinity of the chosen ligands. Possible use of the simulation results in further construction of Markov state models is also discussed.
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