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

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 (pKa of 6.2) for TSA formation from pH 7.0–7.7 in wild-type GCL. This pKa is similar to the pKa of 6.1 for the 1′,4′-iminopyrimidine (IP)–4′-aminopyrimidinium (APH+) 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 synthes...