Publications

• Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway.

  Meyer E., Leonard N.J., Bhat B., Stubbe J., Smith J.M.

  Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be res ... >> More

  Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The Km for HCO3-in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi. No dependence on the concentration of HCO3-could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established. << Less

  Biochemistry 31:5022-5032(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily.

  Thoden J.B., Kappock T.J., Stubbe J., Holden H.M.

  Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulte ... >> More

  Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2.1 and 2.5 A resolution, respectively. PurK belongs to the ATP grasp superfamily of C-N ligase enzymes. Each subunit of PurK is composed of three domains (A, B, and C). The B domain contains a flexible, glycine-rich loop (B loop, T123-G130) that is disordered in the sulfate-PurK structure and becomes ordered in the MgADP-PurK structure. MgADP is wedged between the B and C domains, as with all members of the ATP grasp superfamily. Other enzymes in this superfamily contain a conserved Omega loop proposed to interact with the B loop, define the specificity of their nonnucleotide substrate, and protect the acyl phosphate intermediate formed from this substrate. PurK contains a minimal Omega loop without conserved residues. In the reaction catalyzed by PurK, carboxyphosphate is the putative acyl phosphate intermediate. The sulfate of the sulfate ion-liganded PurK interacts electrostatically with Arg 242 and the backbone amide group of Asn 245, components of the J loop of the C domain. This sulfate may reveal the location of the carboxyphosphate binding site. Conserved residues within the C-terminus of the C domain define a pocket that is proposed to bind AIR in collaboration with an N-terminal strand loop helix motif in the A domain (P loop, G8-L1). The P loop is proposed to bind the phosphate of AIR on the basis of similar binding sites observed in PurN and PurE and proposed in PurD and PurT, four other enzymes in the purine pathway. << Less

  Biochemistry 38:15480-15492(1999) [PubMed] [EuropePMC]

• N5-carboxyaminoimidazole ribonucleotide: evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli.

  Mueller E.J., Meyer E., Rudolph J., Davisson V.J., Stubbe J.

  Conversion of aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins, PurE and PurK, previously thought to be subunits of a single enzyme, AIR carboxylase. Past studies revealing an ATP requirement for this reaction (Meyer et ... >> More

  Conversion of aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins, PurE and PurK, previously thought to be subunits of a single enzyme, AIR carboxylase. Past studies revealing an ATP requirement for this reaction (Meyer et al., 1992), in conjunction with present studies, reveal that PurE and PurK possess independent catalytic activities. PurK is shown, by NMR spectroscopy, to catalyze the conversion of AIR in the presence of HCO3- and ATP to ADP, P(i), and the carbamate of AIR (designated N5-CAIR). PurE has been shown by NMR spectroscopy and kinetic analysis, to catalyze the reversible conversion of N5-CAIR and CAIR. N5-CAIR has a half-life of 0.9 min at pH 7.8 and 30 degrees C. Thus, two new enzymatic activities and a new intermediate have been discovered in the de novo purine biosynthetic pathway of E. coli. << Less

  Biochemistry 33:2269-2278(1994) [PubMed] [EuropePMC]