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J Stubbe

Publications and source records attributed to J Stubbe.

At least 73 records · Page 4Linked to original sources
PubMed1994

Coenzyme B12-dependent ribonucleotide reductase: evidence for the participation of five cysteine residues in ribonucleotide reduction.

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the conversion of ribonucleotides to 2'-deoxyribonucleotides and requires adenosylcobalamin (AdoCbl) as a cofactor. Recent cloning, sequencing, and expression of this protein [Booker, S., & Stubbe, J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8352-8356] have now allowed its characterization by site-directed mutagenesis. The present study focuses on the role of five cysteines postulated to be required for catalysis. The choice of which of the ten cysteines of RTPR were to be mutated was based on extensive studies on the Escherichia coli ribonucleoside diphosphate reductase. Despite the differences between these two reductases in primary sequence, quaternary structure, and cofactor requirements, their mechanisms are strikingly similar. The mutagenesis studies reported herein further suggest that the complex role of the five cysteines is also very similar. A variety of single and double mutants of RTPR were prepared (C731S, C736S, C731 and 736S, C119S, C419S, C408S, and C305S), and their interaction with the normal substrate (CTP) was characterized under several sets of conditions. Mutants C731S, C736S, and C731 and 736S all catalyzed the formation of dCTP at rates similar to those of the wild-type (wt) enzyme in the presence of the artificial reductant DTT. In the presence of the in vivo reducing system (thioredoxin, thioredoxin reductase, and NADPH), however, each of these mutants catalyzed the formation of only 0.6-0.8 dCTPs per mole of enzyme. The inability of these mutants to catalyze multiple turnovers with respect to the in vivo reducing system suggests that their function might be to transfer reducing equivalents from thioredoxin into the active site disulfide of the reductase. Mutants C119S and C419S were targeted as being the active site cysteines, the ones which directly reduce the ribonucleotide substrate. As expected, neither of these mutants catalyzed the formation of dCTP. However, they did catalyze a time-dependent formation of cytosine, destruction of the cofactor, and the appearance of a chromophore associated with the protein--all phenotypes previously observed for the corresponding active site cysteines of the E. coli reductase. Mutant C408S was unable to catalyze dNTP production or cytosine release. Moreover, it was ineffective in catalyzing two additional reactions which are unique to this enzyme: the exchange of tritium from the 5' hydrogens of AdoCbl with H2O and the destruction of AdoCbl under anaerobic conditions to give 5'-deoxyadenosine and cob(II)alamin. These results are consistent with the role of this cysteine as the protein radical responsible for initiating catalysis.

Allosteric Regulation↗
PubMed1994

Reactions catalyzed by 5-aminoimidazole ribonucleotide carboxylases from Escherichia coli and Gallus gallus: a case for divergent catalytic mechanisms.

A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E. coli and G. gallus has been conducted using in vivo and in vitro studies. In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. The E. coli PurE and the C-terminal domain of the G. gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities. The substrate requirements of AIR carboxylases from E. coli and G. gallus have been compared by a series of in vitro experiments. The carbamic acid, N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) is a substrate for the E. coli PurE (Mueller et al., 1994) but not for the G. gallus AIR carboxylase. In contrast, AIR and CO2 are substrates for the G. gallus AIR carboxylase. The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5- aminoimidazole ribonucleotide (NAIR). NAIR is a tight-binding inhibitor of the G. gallus AIR carboxylase (K(i) = 0.34 nM) but only a steady-state inhibitor (K(i) = 0.5 microM) of the E. coli PurE. These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoimidazole Carboxamide↗
PubMed1994

Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity.

Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1. The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process. A mechanistic model has been proposed for the polymerization process [Griebel, R., Smith, Z., & Merrick, J. (1968) Biochemistry 7, 3676-3681] in which two cysteines are required for catalysis. This model is based on the well-characterized enzymes involved in fatty acid biosynthesis. To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein. CD and antibody studies suggest that the mutant proteins are properly folded. The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for synthase activity as well. This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [3H]-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyltransferases↗
PubMed1994

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

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.

Adenosine Diphosphate↗
PubMed1993

A non-heme iron protein with heme tendencies: an investigation of the substrate specificity of thymine hydroxylase.

Thymine hydroxylase from Rhodotorula glutinis catalyzes the oxidation of thymine to its alcohol, aldehyde, and carboxylic acid in three successive reactions. Each step involves stoichiometric consumption of O2 and alpha-ketoglutarate and formation of CO2 and succinate. Given the promiscuity of this enzyme, it was hoped that it would serve as a prototype for understanding the mechanism of this class of enzymes, the non-heme Fe2+ dioxygenases. Kinetic parameters for thymine, O2, Fe2+, and alpha-ketoglutarate have been determined, and isotope effect analysis of (trideuteriomethyl)thymine with enzyme reveals D(V) = 2.08 and D(V/K) = 1.11 at saturating O2. The kinetic parameters for (hydroxymethyl)uracil oxidation have been determined, and incubation of (5'-R)- and (5'-S)-[5'-2H]-5-(hydroxymethyl)uracil with enzyme reveals stereospecific removal of the pro-S hydrogen. No apparent isotope effect is observed in this reaction. The substrate specificity of this enzyme has been examined in detail. The enzyme can catalyze epoxidation, oxidation of a thioether to a sulfoxide and a sulfone, hydroxylation of an unactivated carbon-hydrogen bond, and oxidation of a methylamine to formaldehyde, as revealed through studies with 5-vinyluracil, 5-(methylthio)uracil, 5,6-dihydrothymine, and 1-methylthymine, respectively. In each case, the products were identified by gas chromatography-mass spectrometry, and 18O2-labeling studies revealed that one atom from O2 is incorporated into each product. The enzyme has also been shown to catalyze an uncoupling of hydroxylation and decarboxylation in the presence of a substrate analog incapable of undergoing hydroxylation or a substrate that is difficult to oxidize.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Mechanism-based inactivation of thymine hydroxylase, an alpha-ketoglutarate-dependent dioxygenase, by 5-ethynyluracil.

5-Ethynyluracil was shown to be a mechanism-based inactivator of thymine 7-hydroxylase, with Ki = 22 microM and a k2 = 2.6 min-1l Inactivation resulted in covalent modification of the enzyme with a stoichiometry of approximately 1 adduct/enzyme molecule. The reaction of thymine 7-hydroxylase with 5-ethynyluracil also generated two products: 5-carboxyuracil and uracil-5-acetylglycine. The enzyme adduct was stable at pH 2, 8, and 10 and stable to treatment with hydroxylamine. Following trypsin digestion of labeled enzyme, two labeled peptides corresponding to 45% of the adduct were isolated and sequenced. The results demonstrated the presence of a single modified amino acid. Tandem mass spectrometry suggested that the modified amino acid is tyrosine, which is linked to the inhibitor in an unprecedented fashion.

Amino Acid Sequence↗
PubMed1993

Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate.

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, and catalyzes the conversion of nucleotides to deoxynucleotides. The mechanism of inactivation of RDPR by 2'-azido-2'-deoxynucleoside 5'-diphosphate (N3UDP) has been examined using a variety of isotopically labeled derivatives: (1'-, 2'-, 3'-, or 4'-[2H])-N3UDPs and 2'-[15N3, 13C]-N3UDP. Electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopy studies using these compounds indicate that the 2' carbon-nitrogen bond to the azide moiety is cleaved prior to or upon formation of the nitrogen-centered radical derived from the azide moiety of N3UDP. EPR studies reveal no hyperfine interactions of the nitrogen-centered radical with the 1', 2', 3', or 4' hydrogens of N3UDP. ESEEM studies however, reveal that the 1' and 4' deuterons are 3.3 +/- 0.2 and 2.6 +/- 0.3 A, respectively, from the nitrogen-centered radical. Further support for carbon-nitrogen bond cleavage is derived from studies of the interaction of oxidized R1, C225SR1, and C462SR1 with R2 and N3UDP. In all three cases, in contrast to the results with the wild type R1, azide is detected. Nitrogen-centered radical is not observed with either oxidized R1 or C225SR1 but is observed with C462SR1. These results suggest that C225 is required for the conversion of azide into N2 and a nitrogen-centered radical. The dynamics of the inactivation of RDPR by N3UDP have also been examined. Use of [3'-2H]N3UDP reveals an isotope effect of approximately 2 on the loss of the tyrosyl radical and the rate of inactivation of RDPR. In both cases the kinetics are complex, suggesting multiple modes of inactivation. In addition, several modes of inactivation are required to explain the observation that loss of the tyrosyl radical is slower than the rate of inactivation. Studies using [5'-3H]N3UDP reveal that the rapid inactivation is the result of the formation of a tight noncovalent complex between modified nucleotide, nitrogen-centered radical and RDPR. Destruction of the nitrogen-centered radical is a slow process which appears to be accompanied by decomposition of the modified nucleotide into PPi, uracil, and 2-methylene-3(2H)-furanone. The latter covalently modifies R1 and ultimately leads to loss of approximately 50% of the activity of R1.

Azides↗
PubMed1993

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii.

Amino Acid Sequence↗
PubMed1993

Isotope effects on the cleavage of DNA by bleomycin: mechanism and modulation.

Sequence-specific isotope effects on the cleavage of DNA at thymidine residues by activated iron.bleomycin (Fe.BLM) have been observed upon incorporation of [4'-2H]thymidine into the DNA. The effects may be quantitated by end-labeling the DNA with phosphorus-32, generating a sequence ladder by gel electrophoresis, and measuring the relative damage at cleavage sites by autoradiography or phosphorimager technology. Results with DNA deuteriated at other positions of the deoxyribose ring, such as, the 5'- or 2'-carbon, afford no isotope effect suggesting high regiospecificity for the BLM-mediated carbon-hydrogen bond cleavage. The magnitude of the 4'-isotope effects for Fe.BLM ranges from 2 to 7 (+/- 0.3) and is not sensitive to changes in the partition ratio of the two proposed cleavage pathways. However, the isotope effect is sensitive to structural changes in BLM, such as with tallysomycin, and to changes in metal and cleavage mechanism, such as with Co.BLM. The variability of the effects are discussed in light of the factors responsible for the observation of V/K isotope effects. The findings suggest that the kinetics, thermodynamics, and geometry of carbon-hydrogen bond cleavage by Fe.BLM permit the observation of large isotope effects on this chemical step. Fe.BLM may be viewed, therefore, as a special case.

Base Sequence↗
PubMed1993

Ribonucleotide reductases: radical enzymes with suicidal tendencies.

Ribonucleotide reductases isolated from E. coli and from L. leichmannii differ considerably in their primary and quaternary structures, as well as in their cofactor requirements. Despite these differences, studies with the wt enzymes and the normal substrate, and with the wt enzymes and a variety of mechanism-based inhibitors, demonstrate amazing mechanistic similarities between the two reductases. Recent studies with five cysteine mutants of both reductases reveal strikingly similar phenotypes, indicating that, despite the differences in the primary structures, the groups involved in catalysis in both enzymes appear to be similar.

Amino Acid Sequence↗
PubMed1993

Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules.

Antibodies raised against the Alcaligenes eutrophus poly-beta-hydroxybutyrate (PHB) synthase polypeptide were used for immunocytochemical localization of the synthase enzyme in whole cells and purified PHB granules. The data presented demonstrate for the first time that the synthase enzyme is located on the surface of the PHB granule rather than being incorporated inside the granule during its formation. From these basic observations and data from the recent literature, a model of granule assembly is proposed.

Acyltransferases↗
PubMed1992

A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing.

Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Multiple cysteins have been postulated to play a key role in this process. To test the role of various cysteines in nucleotide reduction, a variety of single and double mutants of the R1 subunit were prepared: C754S, C759S, C754-759S, C462S, C462A, C230S, and C292S. Due to the expression system, each mutant contains small amounts of contaminating wt-R1 (estimated to be 1.5-3% based on activity). An epitope tagging method in conjunction with anion exchange chromatography was used to partially resolve the mutant R1 from the wt-R1. The interaction of these mutants with the normal substrate was studied, which allowed a model to be proposed in which five cysteines of the R1 subunit of RDPR play a role in catalysis. C754S and C759S R1s catalyze CDP formation at rates similar to wt-R1 when DTT is used as a reductant. However, when thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH is used as reductant, the rates of dNDP production are similar to those expected for contaminating wt-R1 present as a heterodimer with the mutant. The impaired nature of these mutants with respect to reduction by TR suggests that their function is to transfer reducing equivalents from TR to the active site disulfide of R1 produced during NDP reduction. Single-turnover experiments, designed to avoid the problem of contaminating wt-R1, also support this role for C754 and C759. The double serine mutant of 754 and 759 has catalytic activity with DTT that is one-third the rate of wt-R1 with thioredoxin. C225 and C462 are thought to be the active site cysteines oxidized concomitantly with NDP reduction. Conversion of these cysteines to serines results in R1 mutants which convert the normal substrate into a mechanism-based inhibitor. C462SR1 upon incubation with R2 and [3'-3H,U-14C]UDP results in uracil release, 3H2O production, 3H,14C-labeled protein which has an absorbance change at 320 nm, and slow loss of the tyrosyl radical on R2. The isotope effect (kH/k3H) on 3' carbon-hydrogen bond cleavage is 1.7. This sequence of events is independent of the reductant, consistent with the postulate that C462 is an active site thiol. The C462AR1 has properties similar to C462SR1. Several additional mutant R1s, C230SR1, and C292SR1 were shown to have activities similar to wt-R1 with both TR/TRR/NADPH and DTT.

Base Sequence↗
PubMed1992

Interaction of C225SR1 mutant subunit of ribonucleotide reductase with R2 and nucleoside diphosphates: tales of a suicidal enzyme.

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3'-3H,U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/k3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H2O, indicative of 3' carbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3'-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the R1 polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of R1, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis↗
PubMed1992

Characterization of C439SR1, a mutant of Escherichia coli ribonucleotide diphosphate reductase: evidence that C439 is a residue essential for nucleotide reduction and C439SR1 is a protein possessing novel thioredoxin-like activity.

Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Cysteine 439 is proposed to be the protein radical on R1 which initiates the reduction reaction by cleavage of the 3' carbon-hydrogen bond of the nucleotide (Mao et al., 1992a,b). C439 is thus proposed to be essential for catalysis. The C439S mutant of R1 (C439SR1) was prepared. The structure of this mutant was determined to be similar to wt-R1, based on identical CD spectra, isolation via an affinity column specific for the allosteric binding domain, binding of the substrate GDP, and competition with R1 for binding to R2. Preparations of C439SR1 are contaminated with low levels of wt-R1 due to the expression system. The wt-R1 in these preparations can be specifically inactivated by the stoichiometric mechanism-based inhibitor, 2'-azido-2'-deoxyuridine 5'-diphosphate. The activity of the resulting C439SR1 was shown to be less than 0.03% that of the corresponding wt-R1. This is the lower limit of detection with the present assay method. Thus C439 appears to be essential for catalysis. During these studies an unexpected activity of the C439SR1 was uncovered. Its additional cysteines, presumably C754 and C759, appear to function as a thioredoxin with the wt-R1, even though it is incapacitated with respect to nucleotide reduction.

Base Sequence↗
PubMed1992

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.

Bleomycin↗
PubMed1992

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

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.

Adenosine Triphosphatases↗