Abstract:
:Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Tittmann K,Golbik R,Uhlemann K,Khailova L,Schneider G,Patel M,Jordan F,Chipman DM,Duggleby RG,Hübner Gdoi
10.1021/bi034465osubject
Has Abstractpub_date
2003-07-08 00:00:00pages
7885-91issue
26eissn
0006-2960issn
1520-4995journal_volume
42pub_type
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