Thiamine is converted to the active ThDP by direct pyrophosphate transfer from ATP using thiamine diphosphoki-nase (EC, which is regulated through a riboswitch; a conformational transition in the RNA transcripts encoding the synthetic enzyme that is induced by the binding of the ThDP itself.143 ThDP performs important metabolic functions by assisting in making or breaking bonds between carbon and sulfur, oxygen, hydrogen, nitrogen, and carbon. Thus, it plays the central role in several important anabolic and catabolic intermediary pathways (see Table 28.9), such as glycololysis, the citric acid cycle, and amino acid metabolism. Broadly all these enzymes are categorized as decar-boxylase- or transferase-type enzymes based on their mechanism of action; all share certain mechanistic similarities as shown in Figure 28.24.143

The whole catalytic cycle can be divided into three steps: activation, first half-reaction, and the second half-reaction. The C2 carbon of ThDP is first activated by deprotonation forming a nucleophilic ylide. ThDP first tautomerizes into an unusual imino form, and the nitrogen atom of the imine then abstracts the proton. Overall, the conformation of ThDP is maintained by the enzymes so as to favor activation by positioning N4' of the amino pyrimidine group in close proximity to the C2 of the thiazolium ring. The first half-reaction involves nucleophilic attack on a substrate carbonyl cleaving the substrate (e.g., pyruvate for decarboxylase and a ketose sugar for transketolase) to release the first product—CO2 for decarboxylases, and an aldose with two less carbons for transketolase—leaving behind an enamine complex. This is common to all types of ThDP-dependent enzymatic reactions. The second half-reaction is specialized for each category of ThDP-dependent enzyme, which involves the nucleo-philic attack of the enamine intermediate on the incoming second substrate to release the final product. In case of pyru-vate or paralogous decarboxylases, this second substrate is a proton producing an aldehyde and releasing the ThDP, whereas in the case of transketolase, this second substrate is an aldose sugar producing a ketose sugar with two more carbon atoms and releasing the ThDP.143

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