Experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations.
Thiamin diphosphate (ThDP), the vitamin B1 coenzyme, is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/C2-carbanion of the thiazolium ring and the C2alpha-carbanion (or enamine) once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state (X-ray) methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of a several intermediates which are stable under acidic conditions, and circular dichroism detection of the 1',4'-imino tautomer of ThDP in some of the intermediates. Some of these methods also enable the investigator to determine the rate-limiting step in the complex series of steps.
Jordan
Frank
F
Department of Chemistry, Rutgers University, Newark, NJ 07102, USA.
Nemeria
Natalia S
NS
D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism*
D003067:Coenzymes; D004798:Enzymes; D009097:Multienzyme Complexes; D013835:Thiamine Pyrophosphate