Glyoxylate Carboligase:
A Unique Thiamin Diphosphate-Dependent
Enzyme That Can Cycle between the 4′-Aminopyrimidinium and
1′,4′-Iminopyrimidine Tautomeric Forms in the Absence
of the Conserved Glutamate
- Publication date
- Publisher
Abstract
Glyoxylate carboligase (GCL) is a thiamin diphosphate
(ThDP)-dependent
enzyme, which catalyzes the decarboxylation of glyoxylate and ligation
to a second molecule of glyoxylate to form tartronate semialdehyde
(TSA). This enzyme is unique among ThDP enzymes in that it lacks a
conserved glutamate near the N1′ atom of ThDP (replaced by
Val51) or any other potential acid–base side chains near ThDP.
The V51D substitution shifts the pH optimum to 6.0–6.2 (p<i>K</i><sub>a</sub> of 6.2) for TSA formation from pH 7.0–7.7
in wild-type GCL. This p<i>K</i><sub>a</sub> is similar
to the p<i>K</i><sub>a</sub> of 6.1 for the 1′,4′-iminopyrimidine
(IP)–4′-aminopyrimidinium (APH<sup>+</sup>) protonic
equilibrium, suggesting that the same groups control both ThDP protonation
and TSA formation. The key covalent ThDP-bound intermediates were
identified on V51D GCL by a combination of steady-state and stopped-flow
circular dichroism methods, yielding rate constants for their formation
and decomposition. It was demonstrated that active center variants
with substitution at I393 could synthesize (<i>S</i>)-acetolactate
from pyruvate solely, and acetylglycolate derived from pyruvate as
the acetyl donor and glyoxylate as the acceptor, implying that this
substitutent favored pyruvate as the donor in carboligase reactions.
Consistent with these observations, the I393A GLC variants could stabilize
the predecarboxylation intermediate analogues derived from acetylphosphinate,
propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric
forms notwithstanding the absence of the conserved glutamate. The
role of the residue at the position occupied typically by the conserved
Glu controls the pH dependence of kinetic parameters, while the entire
reaction sequence could be catalyzed by ThDP itself, once the APH<sup>+</sup> form is accessible