Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.
The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.
Zhang
Sheng
S
Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.
Zhou
Leon
L
Nemeria
Natalia
N
Yan
Yan
Y
Zhang
Zhen
Z
Zou
Yu
Y
Jordan
Frank
F
D000107:Acetylation; D041961:Deuterium Exchange Measurement; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011522:Protons; D011768:Pyruvate Dehydrogenase Complex; C453599:pyruvate dehydrogenase multienzyme complex E1; C047467:2-(1-hydroxyethyl)thiamine pyrophosphate; D013835:Thiamine Pyrophosphate