1 research outputs found
Elucidation of the Catalytic Mechanism of a Miniature Zinc Finger Hydrolase
To improve our mechanistic
understanding of zinc metalloenzymes,
we report a joint computational and experimental study of a minimal
carbonic anhydrase (CA) mimic, a 22-residue Zn-finger hydrolase. We
combine classical molecular dynamics (MD) simulations, quantum mechanics/molecular
mechanics (QM/MM) geometry optimizations, and QM/MM free energy simulations
with ambient and high-pressure kinetic measurements to investigate
the mechanism of the hydrolysis of the substrate <i>p</i>-nitrophenylacetate (pNPA). The zinc center of the hydrolase prefers
a pentacoordinated geometry, as found in most naturally occurring
CAs and CA-like enzymes. Two possible mechanisms for the catalytic
reaction are investigated. The first one is analogous to the commonly
accepted mechanism for CA-like enzymes: a sequential pathway, in which
a Zn<sup>2+</sup>-bound hydroxide acts as a nucleophile and the hydrolysis
proceeds through a tetrahedral intermediate. The initial rate-limiting
step of this reaction is the nucleophilic attack of the hydroxide
on pNPA to form the tetrahedral intermediate. The computed free energy
barrier of 18.5 kcal/mol is consistent with the experimental value
of 20.5 kcal/mol obtained from our kinetics experiments. We also explore
an alternative reverse protonation pathway for the hydrolase, in which
a nearby hydroxide ion from the bulk acts as the nucleophile (instead
of a zinc-bound hydroxide). According to QM/MM MD simulations, hydrolysis
occurs spontaneously along this pathway. However, this second scenario
is not viable in our system, as the tertiary structure of the hydrolase
lacks a suitably positioned residue that would act as a general base
and generate a hydroxide ion from a nearby bulk water molecule. Hence,
our combined theoretical and experimental study indicates that the
investigated minimal CA mimic retains the essential mechanistic features
of CA-like enzyme catalysis. The high-pressure experiments show that
its catalytic efficiency can be enhanced by applying hydrostatic pressure.
According to the simulations, more drastic improvements might be afforded
by mutations that make the reverse protonation pathway accessible