jp212186q_si_001.pdf (1.29 MB)
Thermodynamic and Kinetic Stabilities of Active Site Protonation States of Class C β‑Lactamase
journal contribution
posted on 2012-04-26, 00:00 authored by Ravi Tripathi, Nisanth N. NairBy employing computationally intensive molecular dynamics
simulations
using hybrid quantum–mechanical/molecular–mechanical
approach, we analyze here the kinetic and thermodynamic stabilities
of various active site protonation states of a fully solvated class
C β‑lactamase. We report the detailed mechanism of proton
transfer between catalytically important active site residues and
the associated free energy barriers. In the apoenzyme, significant
structural changes are associated with the proton transfer, and the
orientations of active site residues are distinctly different for
various protonation states. Among several propositions on the protonation
state of the apoprotein, we find that the one with Tyr150 deprotonated and both Lys67 and Lys315 residues
being protonated is the most stable one, both thermodynamically and
kinetically. However, the equilibrium structure at room temperature
is a dynamic one, with Lys315Hζ delocalized
between Tyr150Oη and Lys315Nζ. Of great importance, the kinetic and thermodynamic
stability of protonation states are significantly affected on noncovalently
complexing with cephalothin, an antibiotic molecule. The equilibrium
structure of the enzyme–substrate (precovalent) complex has
a dynamic protonation state where a proton shuttles frequently between
the Tyr150Oη and Lys67Nζ. We examine here the genesis of the manifold change
in stability at the molecular level. The importance of our observations
toward understanding the reactivity of the enzyme is discussed and
experimental observations are rationalized.