NMR analysis of covalent intermediates in thiamin diphosphate enzymes.

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

Biochemistry

journal_title

Biochemistry

authors

Tittmann K,Golbik R,Uhlemann K,Khailova L,Schneider G,Patel M,Jordan F,Chipman DM,Duggleby RG,Hübner G

doi

10.1021/bi034465o

subject

Has Abstract

pub_date

2003-07-08 00:00:00

pages

7885-91

issue

26

eissn

0006-2960

issn

1520-4995

journal_volume

42

pub_type

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