Structural and Kinetic Analysis of Catalysis by a Thiamin Diphosphate-Dependent Enzyme, Benzoylformate Decarboxylase
Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active...
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Published in | Biochemistry (Easton) Vol. 42; no. 7; pp. 1820 - 1830 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
25.02.2003
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Subjects | |
Online Access | Get full text |
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Summary: | Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K m of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K i of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k cat of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K m and k cat. Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-α-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-α-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction. |
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Bibliography: | This research was supported at Purdue University by NSF Grant 9733552-MCB and by David and Lucille Packard Foundation Fellowship 99-1463 (to M.S.H.), by NIH Grant GM-50380, by the Rutgers Busch Biomedical Fund, by the Rutgers Board of Governors Fund, by Roche Diagnostics Inc. (Indianapolis, IN), and by NSF Grant BIR94/13198 (to F.J.), and by NIH Grant GM-40570 (to G.L.K.). ark:/67375/TPS-MVSXDB0P-G istex:B4A05EFE7CDC181E20EB540D1261FF745B9BC931 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi026490k |