Investigation of thiamin diphosphate-dependent catalysis by benzoylformate decarboxylase
Abstract
Benzoylformate decarboxylase (BFD) is a ThDP-dependent enzyme that catalyzes a non-oxidative conversion of benzoylformate into benzaldehyde and carbon dioxide. BFD is one of the four ThDP-dependent enzymes with the known crystal structure, the other three being pyruvate decarboxylase (PDC), pyruvate oxydase (POX) and transketolase (TK). In spite of extensive studies of this metabolically important enzyme group, the chemical mechanism of ThDP-dependent catalysis and the catalytic roles of the active-site residues of the group's members have not yet been completely elucidated. Analysis of the four active-site mutants of BFD (S26A, E28A, H70A, and H281A) is reported. Steady-state kinetic analysis shows that all of these residues are important in catalysis. Stopped-flow experiments show that His 70, Glu 28, and Ser 26 are involved in the steps up to and including decarboxylation. Ser 26 and His 281 are involved in product release. An improved model of the protonation/deprotonation events during the BFD catalytic cycle is proposed. Crystal structures of the WT BFD- R-mandelate and H70A BFD-p-nitrobenzoylformate complexes have been solved. The structure suggests that the serine residue (Ser 26) in the active site of BFD would form a hydrogen bond with a carboxyl group of the substrate, while His 70 hydrogen bonds to the Cα hydroxyl group. None of the active-site aromatic residues interact with the benzyl ring. A novel competitive inhibitor and inactivator of BFD, benzoylphosphonate, has been discovered. A 3.3 Å crystal structure of benzoylphosphonate-inactivated BFD shows that Ser 26 is modified. Mass-spectroscopic analysis of the modified BFD confirmed phosphorylation of this residue. A possible chemical mechanism of phosphorylation of Ser 26 by benzoylphosphonate, which involves one of the active site histidine residues, is proposed.
Degree
Ph.D.
Advisors
Hasson, Purdue University.
Subject Area
Biochemistry|Biophysics
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