Catalyzing separation of carbon dioxide in thiamin diphosphate‐promoted decarboxylation

Abstract

Decarboxylases and intermediatesThiamin diphosphate (ThDP) is a cofactor that promotes the decarboxylation of 2-ketoacids through formation of covalent derivatives between its C2 thiazolium and the carbonyl of the substrate. Combination of a protein and ThDP in a holoenzyme provides substrate specificity and the general enzymic advantage of reduced translational entropy that favors addition processes [1,2]. The covalent intermediate undergoes cleavage of a bond to a carboxylate group derived from the 2-ketoacid, resulting in production of carbon dioxide. This also produces a residual acyl anion equivalent [3-6] with a delocalized structure that can also be represented as a neutral enamine. The sequence is illustrated for the decarboxylation of pyruvic acid by pyruvate decarboxylases in Scheme 1.Protonation at the basic carbon and elimination of ThDP leads to formation of an aldehyde (giving a net substitution of a proton for carbon dioxide). Oxidation of the same intermediate would yield an acid, while reaction with a carbonyl carbon gives a condensation product. The general route is based on concepts originally developed by Breslow [7][8][9] based on studies of model compounds related to ThDP. Details of reaction patterns within that pathway reveal previously unrecognized aspects of enzymic catalysis [3,10].Synthetic analogs of the covalent intermediates have been prepared and studied in order to arrive at a quantitative understanding of the separate functions of the cofactor and protein [11,12]. Spectroscopic analysis of the conjugates of thiamin and ketoacids has enabled specific and quantitative identification of the coenzyme derivatives bound to proteins in enzymic reactions [13][14][15].

Double Duty for a Conserved Glutamate in Pyruvate Decarboxylase: Evidence of the Participation in Stereoelectronically Controlled Decarboxylation and in Protonation of the Nascent Carbanion/Enamine Intermediate,

“…Such a mechanism has been proposed for other decarboxylating enzymes, e.g., OMDCase (38). Also, because variant Glu473Gln is apparently defective in protonating the carbanion/enamine intermediate but still efficiently catalyzes decarboxylation of LThDP, it appears unlikely that carbanion protonation by Glu473 drives decarboxylation of LThDP by preventing an internal return of CO 2 as previously shown in model reactions (39, 40). …”

Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

“…Due to the extremely high conservation scores of the examined seven residues (Figure 5A), all the Ala substituted mutants lost their original activities. According to a reported study of other amino acid decarboxylases, the carbon dioxide releasing process from the α-carbon of the substrate is the ratelimiting step of decarboxylation reactions (Kluger and Rathgeber, 2008); thus, the residues near the carboxyl group of Arg where its dynamic interaction occurs were selected for further Ala scanning. Although mutating the substrate interacting residues is very likely to reduce the original activity of the enzyme (Toscano et al, 2007), interestingly enough, a mutant with improved activity was discovered.…”

Computational screening of novel thiamine-catalyzed decarboxylation reactions of 2-keto acids

“…Experimental [30] and theoretical studies [31][32][33][34][35] have been aimed at understanding the mechanism of the reaction and the structures of the intermediates associated with the reaction. The first step of the pathway can be written as Pyruvic acid þ CoA þ ferredoxin ðoxÞ $ acetyl-CoA þ CO 2 þ ferredoxin ðredÞ ð 1Þ…”