NMR analysis of covalent intermediates in thiamin diphosphate enzymes.
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.
Tittmann
Kai
K
Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie, Kurt-Mothes-Strasse 3, D-06099 Halle, Germany. kai@bc.biochemtech.uni-halle.de
Golbik
Ralph
R
Uhlemann
Kathrin
K
Khailova
Ludmila
L
Schneider
Gunter
G
Patel
Mulchand
M
Jordan
Frank
F
Chipman
David M
DM
Duggleby
Ronald G
RG
Hübner
Gerhard
G
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000737:chemistry*; D017355:Zymomonas / Q000201:enzymology*
D011522:Protons; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate