Abstract
Thiamin diphosphate (ThDP), the biologically active form of vitamin B1, is an essential cofactor that is involved in numerous metabolic pathways, such as the oxidative and non-oxidative decarboxylation of 2-oxo acids, interketol transfer between sugar phosphates, electron transfer reactions and the formation of amino acid precursors. ThDP consists of a thiazolium nucleus, an aminopyrimidine ring, and a diphosphate moiety that is required to provide tight binding to the target enzyme. The electrophilic nature of the positivelycharged thiazolium ring is key to the stabilization of carbanion states of the cofactor (i.e. the ThDP ylide) and of covalent substrate-ThDP conjugates in terms of a ‘Umpolung – or polarity inversion’ mechanism predestining ThDP as an effective participant in the reversible catalytic cleavage of carbon-carbon bonds of vicinal dicarbonyl or a-hydroxyketone groups, exhibiting a chemical ability not possessed by protein functional groups. The first committed step in all ThDP-dependent enzymes is the deprotonation of the thiazolium C2 and attack of the resulting carbanion on a substrate carbonyl. X-ray crystallographic analysis of ThDP enzymes has demonstrated the cofactor to be bound in the canonical V conformation, with the amino group of the aminopyrimidine and C2 of the thiazolium moiety juxtaposed with respect to hydrogen-bonding distance. A strictly conserved glutamate residue interacts with nitrogen 1 of the aminopyrimidine. The conserved structure of the cofactor binding site, and the observed loss of activity accompanyingmutagenesis of the conserved glutamate or uponuse of ThDP analogs lacking nitrogen 1 or the amino group of the aminopyrimidine, all support a mechanism in which proton removal fromC2 of ThDP is assisted by a catalytic triad consisting of the conserved glutamate, the aminopyrimidine and thiazolium moieties of the cofactor, presumably involving the imino tautomer of the aminopyrimidineas thegeneralbase forprotonabstraction. This minireview series, based on lectures given at an international conference on thiamin held in Wittenberg, Germany, in May 2008, highlights recent advancements in the mechanistic understanding of ThDP-promoted biochemical reactions. Jordan et al. have accumulated spectroscopic evidence for the occurrence and interconversion of the different tautomeric and ionization states of enzymebound ThDP along the pathways of various ThDP enzymes. The data clearly implicate the delicately balanced equilibria of the different cofactor forms on the enzymes’ active sites, such that all of these states are thermodynamically accessible at the pH optimum of a given ThDP enzyme. As outlined above, a glutamate residue interacting with the cofactor aminopyrimidine has hitherto been believed to be vital for tautomerization of the aminopyrimidine and thus chemical activation of ThDP. However, structural and mechanistic studies on the ThDP-dependent enzyme glyoxylate carboligase (GCL), as reviewed by Shaanan and Chipman, have revealed valine as having replaced the expected glutamate. Mechanistic analysis suggested that GCL sacrifices the otherwise strictly conserved mechanism of protein-cofactor activation as a consequence of evolutionary adaption to avoid formation of overlystabilized intermediates as thermodynamic traps. The oxidative decarboxylation of 2-oxo acids such as pyruvate is a key reaction of intermediary metabolism in virtually all organisms and requires ThDP to act in tandem with additional cofactors (e.g. flavins, iron-sulfur cluster, lipoic acid) as redox partners. The review by Tittmann, devoted to the mechanistic analysis of redox reactions of ThDP enzymes, shows how some of these enzymes make use of elegant radical chemistry in an intimate coupling mechanism of oxidation–reduction and the formation of energy-rich metabolites such as acetyl-CoA or acetyl phosphate.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.