Mechanism and Catalytic Site Atlas

Introduction

Mechanistic studies have shown that the formation of uroporphyrinogen III involves an electrophilic addition of the substrates hydroxymethyl group to C-16 that subsequently results in the cleavage of the C-15 to C-16 bond. This mechanism is referred to as the spiro-mechanism since the key intermediate is a spiropyrrolenine produced by the initial cyclisation. This mechanism is supported by the strong inhibition caused by a spiro-lactam intermediate homologue [PMID:13680202]. Modelling studies with small molecules show that the [1,5]-sigmatropic rearrangement of the substrate is unlikely, whereas fragmentation-recombination should be facile. Thus suggesting that of the various mechanisms suggested for this enzyme, the one shown in here is the most likely [DOI:10.1021/cr00105a009]. The lack of catalytic amino acid residues can be explained due to the fact that mutation of all the surface-exposed and conserved residues (mostly serine and threonine residue), and several of the other residues in the putative active site cleft, did not reveal any single residue that was absolutely required for catalysis [PMID:12196144, PMID:11689424]. This suggests that the residues may function as hydrogen bond donors and acceptors for the variety of carboxylate side chains or pyrrole nitrogens of the substrate. It is also suggested that the enzyme does not make extensive uses of charge-charge interactions with the carboxylate side chains due to the fact that only one of the conserved residues is positively charged. Instead a potentially larger number of weaker interactions may develop between ligand and polar or hydrophobic side chains. The robustness of enzyme activity to mutational analysis may indicate that no one mutation will disrupt substrate binding when a large number of other contacts are left intact [PMID:11689424]. Finally, it has been noted in modelling studies that the active site is larger than the product which suggests that a large conformational change may be required during catalysis [PMID:12196144].