Pyruvate decarboxylase (PDC) catalyzes the nonoxidative decarboxylation of pyruvate into acetaldehyde and carbon dioxide and requires thiamin diphosphate (ThDP) and a divalent cation as cofactors. Recent studies have permitted the assignment of functional roles of active site residues; however, the underlying reaction mechanisms of elementary steps have remained hypothetical. Here, a kinetic and thermodynamic single-step analysis in conjunction with X-ray crystallographic studies of PDC from Zymomonas mobilis implicates active site residue Glu473 (located on the re-face of the ThDP thiazolium nucleus) in facilitating both decarboxylation of 2-lactyl-ThDP and protonation of the 2-hydroxyethyl-ThDP carbanion/enamine intermediate. Variants carrying either an isofunctional (Glu473Asp) or isosteric (Glu473Gln) substitution exhibit a residual catalytic activity of less than 0.1% but accumulate different intermediates at the steady state. Whereas the predecarboxylation intermediate 2-lactyl-ThDP is accumulated in Glu473Asp because of a 3000-fold slower decarboxylation compared to that of the wild-type enzyme, Glu473Gln is not impaired in decarboxylation but generates a long-lived 2-hydroxyethylThDP carbanion/enamine postdecarboxylation intermediate. CD spectroscopic analysis of the protonic and tautomeric equilibria of the cocatalytic aminopyrimidine part of ThDP indicates that an acidic residue is required at position 473 for proper substrate binding. Wild-type PDC and the Glu473Asp variant bind the substrate analogue acetylphosphinate with the same affinity, implying † This work was supported in part by Grant 0126FP/0705M from the Ministry of Education at Saxony-Anhalt and the DFG-funded Göttingen Graduate School for Neurosciences and Molecular Biosciences (both to K.T.). At Rutgers University, the research was supported by National Institutes of Health Grant GM050380 (to F.J.). ‡ The refined model and corresponding structure factor amplitudes have been deposited in the Protein Data Bank of the Research Collaboratory for Structural Biology as entry 3OE1.© American Chemical Society * To whom correspondence should be addressed. Phone: +49-551-3914430. Fax: +49-551-395749. ktittma@gwdg.de. # Current address: Institute for Microbiology and Genetics, Georg-August-University Göttingen, Göttingen, Germany NIH Public Access
Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 September 21.
NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript a similar stabilization of the predecarboxylation intermediate analogue on the enzyme, whereas Glu473Gln fails to bind the analogue. The X-ray crystallographic structure of 2-lactyl-ThDP trapped in the decarboxylation-deficient variant Glu473Asp reveals a common stereochemistry of the intermediate C2α stereocenter; however, the scissile C2α-C(carboxylate) bond deviates bỹ 25-30° from the perpendicular "maximum overlap" orientation relative to the thiazolium ring plane as commonly observed in ThDP enzymes. Because a reactant-state stabiliza...