Carboxylation in de novo purine biosynthesis.

De novo purine biosynthesis is one of two pathways for the synthesis of purine nucleotides that are critical for numerous biological processes, most notably nucleic acid replication. Within the pathway, there is only one carbon-carbon bond formation which is the carboxylation of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Interestingly, there are two unique pathways within purine biosynthesis to accomplish this transformation and this divergence is species specific. In humans and higher eukaryotes, CAIR is synthesized directly from AIR and carbon dioxide by the enzyme AIR carboxylase. In bacteria, yeast, fungi and plants, CAIR synthesis requires two steps. In the first, AIR is converted into the unstable carbamate, N5-CAIR by the enzyme N5-CAIR synthetase. N5-CAIR is then converted into CAIR by transfer of the CO2 group from N5 to C4. This is catalyzed by the enzyme N5-CAIR mutase. This divergence has provided a biochemical rationale for targeting CAIR synthesis in the development of antimicrobial agents, but recent studies have provided strong evidence that AIR carboxylase plays a critical role in several cancers. Given the significance of these enzymes as drug targets, methods to prepare and evaluate these enzymes is of interest. In this chapter, we have accumulated the most relevant assays and provided methods to synthesize the substrates and purify the enzymes.