1 research outputs found
Significance of Individual Residues at the Regulatory Site of Yeast Pyruvate Decarboxylase for Allosteric Substrate Activation
The
catalytic activity of the allosteric enzyme pyruvate decarboxylase
from yeast is strictly controlled by its own substrate pyruvate via
covalent binding at a separate regulatory site. Kinetic studies, chemical
modifications, cross-linking, small-angle X-ray scattering, and crystal
structure analyses have led to a detailed understanding of the substrate
activation mechanism at an atomic level with C221 as the core moiety
of the regulatory site. To characterize the individual role of the
residues adjacent to C221, we generated variants H92F, H225F, H310F,
A287G, S311A, and C221A/C222A. The integrity of the protein structure
of the variants was established by small-angle X-ray scattering measurements.
The analyses of both steady state and transient kinetic data allowed
the identification of the individual roles of the exchanged side chains
during allosteric enzyme activation. In each case, the kinetic pattern
of activation was modulated but not completely abolished. Despite
the crucial role of C221, the covalent binding of pyruvate is not
obligate for enzyme activation but is a requirement for a kinetically
efficient transition from the inactive to the active state. Moreover,
only one of the three histidines guiding the activator molecule to
the binding pocket, H310, specifically interacts with C221. H310 stabilizes
the thiolate form of C221, ensuring a rapid nucleophilic attack of
the thiolate sulfur on C2 of the regulatory pyruvate, thus forming
a regulatory dyad. The influence of the other two histidines is less
pronounced. Substrate activation is slightly weakened for A287G and
significantly retarded for S311A