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Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity
journal contribution
posted on 2012-10-10, 00:00 authored by Jörgen Ådén, Abhinav Verma, Alexander Schug, Magnus Wolf-WatzStructural plasticity is often required for distinct
microscopic
steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AKeco) populates two major
conformations in solution; the open (inactive) and closed (active)
state, and the overall turnover rate is inversely proportional to
the lifetime of the active conformation. Therefore, structural plasticity
is intimately coupled to enzymatic turnover in AKeco. Here,
we probe the open to closed conformational equilibrium in the absence
of bound substrate with NMR spectroscopy and molecular dynamics simulations.
The conformational equilibrium in absence of substrate and, in turn,
the turnover number can be modulated with mutational- and osmolyte-driven
perturbations. Removal of one hydrogen bond between the ATP and AMP
binding subdomains results in a population shift toward the open conformation
and a resulting increase of kcat. Addition
of the osmolyte TMAO to AKeco results in population shift
toward the closed conformation and a significant reduction of kcat. The Michaelis constants (KM) scale with the change in kcat, which follows from the influence of the population of the closed
conformation for substrate binding affinity. Hence, kcat and KM are mutually dependent,
and in the case of AKeco, any perturbation that modulates kcat is mirrored with a proportional response
in KM. Thus, our results demonstrate that
the equilibrium constant of a pre-existing conformational equilibrium
directly affects enzymatic catalysis. From an evolutionary perspective,
our findings suggest that, for AKeco, there exists ample
flexibility to obtain a specificity constant (kcat/KM) that commensurate with
the exerted cellular selective pressure.