Biochemical and mechanistic characterization of Gallus gallus 5-aminoimidazole ribonucleotide carboxylase

Steven Michael Firestine, Purdue University


Only one carbon-carbon bond is formed in de novo purine biosynthesis, namely during the conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). This reaction is catalyzed by the enzyme AIR carboxylase, an unusual enzyme since it carboxylates an aromatic substrate without the aid of cofators. Recently, the enzyme from Escherichia coli has been characterized and it was found that two enzymes are required to accomplish the conversion of AIR to CAIR. The first enzyme, PurK, catalyzed the conversion of AIR to a previously unrecognized purine intermediate $N\sp5$-carbamoyl-5-aminoimidazole ribonucleotide ($N\sp5$-CAIR). $N\sp5$-CAIR is converted to CAIR by the second enzyme AIR carboxylase (purE). This result suggested that de novo purine biosynthesis as classically stated in biochemistry textbooks was incomplete. Our investigations of AIR carboxylase from Gallus gallus (chicken) indicate that this enzyme does not utilize $N\sp5$-CAIR as a substrate but instead directly converts AIR and CO$\sb2$ to CAIR suggesting that the E. coli and G. gallus enzymes, while evolutionarily related, have devised different mechanisms to synthesize CAIR. The mechanism of G. gallus AIR carboxylase is unknown. However, our laboratory has developed the most potent inhibitor known for G. gallus AIR carboxylase. This compound, 4-nitro-5-aminoimidazole riboucleotide (NAIR) is a slow, tight-binding inhibitor of the enzyme with an overall $K\sb{\rm i}$ of 0.34 nM. The slow, tight-binding nature of NAIR has led to the speculation that the electronic character of NAIR is complementary to that displayed by the enzyme for stabilizing the transition state. To investigate this, azole analogs of NAIR and CAIR have been synthesized which display altered electronic properties. All of these compounds bound G. gallus AIR carboxylase with reduced affinity, and the analysis supported the conclusion that the electronic structure of NAIR mimics that of the transition-state. In addition, these data suggest that the enzyme probably proceeds via a tetrahedral intermediate since removal of N3 does not affect binding, whereas removal of the exocyclic amine results in 75,000-fold decreases in binding.




Davisson, Purdue University.

Subject Area

Biochemistry|Organic chemistry|Molecular biology

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