Uroporphyrinogen decarboxylation as a benchmark for the catalytic proficiency of enzymes

Abstract

The magnitude of an enzyme's affinity for the altered substrate in the transition state exceeds its affinity for the substrate in the ground state by a factor matching the rate enhancement that the enzyme produces. Particularly remarkable are those enzymes that act as simple protein catalysts, without the assistance of metals or other cofactors. To determine the extent to which one such enzyme, human uroporphyrinogen decarboxylase, enhances the rate of substrate decarboxylation, we examined the rate of spontaneous decarboxylation of pyrrolyl-3-acetate. Extrapolation of first-order rate constants measured at elevated temperatures indicates that this reaction proceeds with a half-life of 2.3 ؋ 10 9 years at 25°C in the absence of enzyme. This enzyme shows no significant homology with orotidine 5-monophosphate decarboxylase (ODCase), another cofactorless enzyme that catalyzes a very slow reaction. It is proposed that, in both cases, a protonated basic residue (Arg-37 in the case of human UroD; Lys-93 in the case of yeast ODCase) furnishes a counterion that helps the scissile carboxylate group of the substrate leave water and enter a relatively nonpolar environment, stabilizes the incipient carbanion generated by the departure of CO2, and supplies the proton that takes its place.decarboxylase ͉ catalysis ͉ porphyrin ͉ coproporphyrinogen ͉ evolution T he catalytic power of an enzyme can be judged from the rate enhancement that it produces. In general, enzymes act on their substrates at somewhat similar rates, with k cat values ranging between 50 and 5,000 s Ϫ1 . However, the rate constants of the same reactions in the absence of a catalyst vary over a range of at least 15 orders of magnitude. And the rate enhancements produced by enzymes vary over a similarly broad range (Ϸ10 15 -fold), indicating the magnitude of the increase in the enzyme's affinity for the substrate as it passes from the ground state to the transition state (1). Particularly remarkable are those cases in which the enzyme acts as a simple protein catalyst, without the assistance of metals or other cofactors. Such a reaction, the decarboxylation of orotidine 5Ј-phosphate, was found earlier to proceed in the absence of enzyme with a half-time of 7.8 ϫ 10 7 years in the absence of enzyme (2).Here, we describe another reaction, involving a very different substrate (uroporphyrinogen, Uro'gen), that proceeds with a half-life of 2.3 ϫ 10 9 years at 25°C in the absence of enzyme. The enzymes catalyzing these reactions act as pure protein catalysts. Their amino acid sequences show no significant homology.In plants and animals, uroporphyrinogen decarboxylase (UroD, EC 4.1.1.37) catalyzes the first committed step in the biosynthesis of heme, chlorophyll, and the cytochromes. The pioneering experiments of Mauzerall and Granick established that during the action of this single enzyme, the acetate group at the 3-position of each of the 4 pyrrole rings of uroporphyrinogen III (Uro'gen III) is converted to a methyl group, yielding coproporphyrinogen III (Fig. 1) (...

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…These polar residues have been suggested to interact with the propionate side chains of the tetrapyrrole to spatially orient the substrate, although it has been strongly suggested that one of the Arg residues participates in the catalytic cycle. One model proposes that the conserved Asp residue donates a proton to the substrate pyrrole ring adjacent to the acetate side chain (204). This results in decarboxylation, creating an intermediate and requiring that the conserved Arg residue contribute a proton to the methylene group as the ring is deprotonated by the Asp side chain.…”

“…This results in decarboxylation, creating an intermediate and requiring that the conserved Arg residue contribute a proton to the methylene group as the ring is deprotonated by the Asp side chain. Regardless of the exact mechanism, it has been suggested that UROD is a "benchmark" for catalytic proficiency among enzymes without cofactors, with an estimated value for enzyme enhancement of the decarboxylation reactions of ϳ10 17 (204).…”

The Importance of the MM Environment and the Selection of the QM Method in QM/MM Calculations

“…This enzyme performs the quadruple decarboxylation of its substrate URO-III in mere seconds (Bushnell, Erdtman, Llano, Eriksson, & Gauld, 2011). In the absence of this enzyme, the half-life for this same reaction (under standard conditions in solution) is 2.3 billion years (Lewis & Wolfenden, 2008).…”

Protoporphyrin IX: the Good, the Bad, and the Ugly

“…Uroporphyrinogen decarboxylase (EC 4.1.1.37) then removes carboxylic groups from the four acetic acid side chains of uroporphyrinogen III to form coproporphyrinogen III (Lewis and Wolfenden, 2008). This intermediate is then transported into mitochondria through ABCB6 (Krishnamurthy et al, 2006;Krishnamurthy and Schuetz, 2011), where it is decarboxylated to protoporphyrinogen IX by coproporphyrinogen oxidase (EC 1.3.3.3) (Proulx et al, 1993).…”