Effects of nucleotides on N-acetyl-d-glucosamine 2-epimerases (renin-binding proteins): comparative biochemical studies.

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney as a complex of renin, so-called high molecular weight (HMW) renin. Our recent studies demonstrated that human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have purified recombinant human, rat, and porcine RnBPs expressed in Escherichia coli JM 109 cells. The purified recombinant RnBPs existed as dimers and inhibited porcine renin activity strongly. On the other hand, porcine renin inhibited recombinant GlcNAc 2-epimerase activities. The human GlcNAc 2-epimerase activity could not be detected in the absence of a nucleotide, whereas ATP, dATP, ddATP, ADP, and GTP enhanced the human GlcNAc 2-epimerase activity. Other nucleotides had no effect on human GlcNAc 2-epimerase activity. Rat and porcine GlcNAc 2-epimerases were activated by several nucleotides. Nucleotides that enhance the activity of GlcNAc 2-epimerases protect these enzymes against degradation by thermolysin. These results indicate that mammalian RnBPs have GlcNAc 2-epimerase activity and that nucleotides are essential for formation of the catalytic domain of the enzyme.     

Effects of nucleotides on the interaction of renin with GlcNAc 2-epimerase (renin binding protein, RnBP)

Renin binding protein (RnBP), a cellular renin inhibitor, was identified as an enzyme, GlcNAc 2-epimerase. Recombinant RnBP inhibited porcine renin activity in a dose dependent manner. However, the inhibition was neutralized by nucleotides, such as ATP, dATP, dGTP, dCTP or dTTP. Moreover, ATP inhibited the formation of hetero-complex of renin with RnBP, called high molecular weight (HMW) renin. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited the GlcNAc 2-epimerase activity concomitant with the decaying of the dimer to the monomer of the enzyme. The inhibition was modulated in the presence of ATP. These results indicate that nucleotides stabilize the dimeric form RnBP (GlcNAc 2-epimerase) and inhibited the formation of the renin-RnBP hetero complex, HMW renin.     

Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein)

Renin-binding protein (RnBP) is a highly specific renin inhibitor first isolated from porcine kidney. Our recent studies demonstrated that the human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have developed a new assay method for GlcNAc 2-epimerase activity using a system of N-acyl-D-hexosamine oxidase coupled with peroxidase and employed this method to study the effects of renin on GlcNAc 2-epimerase activity. The recombinant human (rh) RnBP existed as a dimer and its GlcNAc 2-epimerase activity was strongly inhibited by the purified renin concomitant with the formation of RnBP-renin heterodimer, so-called high molecular weight (HMW) renin. The renin activity was also inhibited by rhRnBP in a dose-dependent manner. These results indicate that renin is an inhibitor of GlcNAc 2-epimerase, and the renin-RnBP heterodimer HMW renin is an inactive form of both renin and GlcNAc 2-epimerase activities.     

Identification of cysteine-380 as the essential residue for the human N-acetyl-D-glucosamine 2-epimerase (renin binding protein).

Renin binding protein (RnBP) is a proteinous renin inhibitor firstly isolated from porcine kidney. Recently, the protein was identified as the enzyme, N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. The GlcNAc 2-epimerase activity of recombinant human RnBP was specifically inhibited by SH-reagents such as N-ethylmaleimide, 5, 5'-dithiobis-2-nitrobenzoate, and iodoacetic acid, indicating that the most probable reactive site is a cysteine residue. To identify the active site residue(s), we have constructed ten cysteine residue mutants (C41S, C66S, C104S, C125S, C210S, C239S, C302S, C380S, C386S, and C390S) for human GlcNAc 2-epimerase and expressed them in Escherichia coli cells. The relative specific activities of C41S, C66S, C125S, C210S, C239S, C302S, C386S, and C390S are nearly the same to that of the wild-type enzyme. The specific activity of the C104S mutant is 26% of that of the wild-type enzyme. The expression of the C380S mutant in E. coli cells was detected on Western blotting, whereas GlcNAc 2-epimerase activity was not detected in the extract. These results indicate that Cys380 is essential for the enzymatic activity of human GlcNAc 2-epimerase.     

Nonequivalence of the nucleotide binding domains of the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5'-[alpha-(32)P]-triphosphate at 4 degrees C indicate that metalloid stimulation correlates with a >10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with (32)P at the gamma position was used to measure hydrolysis of trinucleotide at 37 degrees C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.     

Studies on the binding of nucleotides by rat brain hexokinase

Various nucleoside di- and triphosphates have been compared with respect to their ability to protect rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activity against inactivation by chymotrypsin, glutaraldehyde, heat, and 5,5′-dithiobis(2-nitrobenzoic) acid. ATP could be distinguished from other nucleoside triphosphates in these comparisons, which may be related to the specificity with which ATP is utilized as a substrate. All nucleoside derivatives examined provided substantial protection against two or more of the above inactivating agents, indicating relatively nonspecific binding of nucleotides by brain hexokinase, consistent with a similar lack of specificity in the inhibition of this enzyme by nucleoside derivatives. The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tetraiodofluorescein (TIF) was enhanced by binding to brain hexokinase. TNS binding was not affected by the presence of various relevant metabolites (Glc, glucose 6-phosphate, ATP), nor did TNS inhibit the enzyme. In contrast, substantial (approximately 70%) decreases in the fluorescence of bound TIF resulted from the addition of various nucleoside derivatives, and TIF served as a competitive inhibitor of brain hexokinase. These observations are consistent with the view that TIF binds to a nucleotide binding site of the enzyme. The inability of nucleotides to totally displace TIF was taken to indicate the existence of an additional TIF binding site (or sites) discrete from the catalytic site, and probably identical to the site(s) at which TNS binds with no effect on catalytic activity. The effects of saturating levels of ATP and ADP were not additive indicating that both compounds were displacing TIF from the same site i.e., a common nucleotide binding site. Glc, mannose, and 2-deoxyglucose greatly enhanced the ability of nucleotides to displace TIF, while fructose, galactose, and N-acetylglucosamine did not, indicating the existence of interactions between hexose and nucleotide binding sites; the hexoses themselves were not effective at displacing TIF. The enhanced binding of nucleotides in the presence of the first three hexoses but not the latter three can be directly correlated with the relative ability of these hexoses to induce specific conformational changes in the enzyme. The hexoses themselves were not effective at displacing TIF. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate could also displace TIF, and as with the nucleotides, a maximum of approximately 70% decrease in fluorescence was observed and the effectiveness of glucose 6-phosphate was enhanced in the presence of Glc. Other hexose 6-phosphates tested were not effective at displacing TIF. The specificity with which hexose 6-phosphates displaced TIF could be correlated with their ability to induce specific conformational change in the enzyme. The results are discussed as they relate to the kinetic mechanism and allosteric regulation by nucleotides that have been proposed for this enzyme.     

Stoichiometry and affinity of nucleotide binding to P-glycoprotein during the catalytic cycle

Drug transport mediated by P-glycoprotein (Pgp) is driven by hydrolysis of ATP at the two cytosolic nucleotide binding domains. However, little is currently known concerning the stoichiometry of nucleotide binding and how both stoichiometry and binding affinity change during the catalytic cycle of the transporter. To address this issue, we used fluorescence techniques to measure both the number of nucleotides bound to P-glycoprotein during various stages of the catalytic cycle and the affinity of nucleotide binding. Results showed that resting state P-glycoprotein bound two molecules of the fluorescent nucleotide derivative, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), whereas the vanadate-trapped transition state bound only one nucleotide molecule. Both resting and transition state P-glycoprotein showed similar affinity for TNP-ATP/TNP-ADP and unlabeled ATP/ADP. Following binding of various drugs, resting state P-glycoprotein displayed a higher affinity for nucleotides, up to 4-fold depending on the compound used. In contrast, the transition state showed substantially lower (up to 3-fold) nucleotide binding affinity when the drug binding site(s) is/are occupied. These results indicate that both nucleotide binding domains of P-glycoprotein are likely to be occupied with either ATP (or ADP) in the resting state and the transition state in the absence of transport substrates. Drugs alter the binding affinity to favor association of ATP with P-glycoprotein at the start of the catalytic cycle and release of ADP from the transition state following nucleotide hydrolysis.     

Binding of ATP to the fructose-2,6-bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase leads to activation of its 6-phosphofructo-2-kinase

To understand the mechanism by which the activity of the 6-phosphofructo-2-kinase (6PF-2K) of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is stimulated by its substrate ATP, we studied two mutants of the enzyme. Mutation of either Arg-279, the penultimate basic residue within the Walker A nucleotide-binding fold in the bisphosphatase domain, or Arg-359 to Ala eliminated the activation of the chicken 6PF-2K by ATP. Binding analysis by fluorescence spectroscopy using 2'(3')-O-(N-methylanthraniloyl)-ATP revealed that the kinase domains of these two mutants, unlike that of the wild type enzyme, showed no cooperativity in ATP binding and that the mutant enzymes possess only the high affinity ATP binding site, suggesting that the ATP binding site on the bisphosphatase domain represents the low affinity site. This conclusion was supported by the result that the affinity of ATP for the isolated bisphosphatase domain is similar to that for the low affinity site in the wild type enzyme. In addition, we found that the 6PF-2K of a chimeric enzyme, in which the last 25 residues of chicken enzyme were replaced with those of the rat enzyme, could not be activated by ATP, despite the fact that the ATP-binding properties of this chimeric enzyme were not different from those of the wild type chicken enzyme. These results demonstrate that activation of the chicken 6PF-2K by ATP may result from allosteric binding of ATP to the bisphosphatase domain where residues Arg-279 and Arg-359 are critically involved and require specific C-terminal sequences.     

The 1.6 A crystal structure of E. coli argininosuccinate synthetase suggests a conformational change during catalysis.

BACKGROUND: Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. RESULTS: The crystal structure of E. coli AS (EAS) has been determined by the use of selenomethionine incorporation and MAD phasing. The structure has been refined at 1.6 A resolution in the absence of its substrates and at 2.0 A in the presence of aspartate and citrulline (EAS*CIT+ASP). Each monomer of this tetrameric protein has two structural domains: a nucleotide binding domain similar to that of the "N-type" ATP pyrophosphatase class of enzymes, and a novel catalytic/multimerization domain. The EAS*CIT+ASP structure clearly describes the binding of citrulline at the cleft between the two domains and of aspartate to a loop of the nucleotide binding domain, whereas homology modeling with the N-type ATP pyrophosphatases has provided the location of ATP binding. CONCLUSIONS: The first three-dimensional structures of AS are reported. The fold of the nucleotide binding domain confirms AS as the fourth structurally defined member of the N-type ATP pyrophosphatases. The structures identify catalytically important residues and suggest the requirement for a conformational change during the catalytic cycle. Sequence similarity between the bacterial and human enzymes has been used for providing insight into the structural and functional effects of observed clinical mutations.     

Src-Abl tyrosine kinase chimeras: replacement of the adenine binding pocket of c-Abl with v-Src to swap nucleotide and inhibitor specificities

Engineered protein kinases with unnatural nucleotide specificity and inhibitor sensitivity have been developed to trace kinase substrate targets. We first engineered unnatural nucleotide specificity into v-Src by mutating one residue, isoleucine 338, to alanine. This position is highly conserved among all kinases in the sense that it is always occupied by either a large hydrophobic residue or threonine. Because of the conservation of this residue and the highly conserved fold of the kinase family, we have attempted to generalize the engineering of all kinases on the basis of our success with v-Src. Although many kinases can be similarly engineered using v-Src as a blueprint, we encountered one kinase, c-Abl, which when mutated, does not display the ability to accept unnatural ATP analogues. To overcome this failure of the engineered c-Abl (T315A) to accept unnatural nucleotides, we developed a new strategy for introducing unnatural nucleotide specificity into kinases. We generated a chimeric kinase in which regions of the kinase domain of c-Abl were swapped with the corresponding regions of v-Src (I338A). Specifically, we engineered two chimeras in which the N-terminal lobe of the SH1 domain of c-Abl was swapped with that of v-Src. These kinase chimeras were found to have the same unnatural nucleotide specificity as that of v-Src (I338A), while retaining the peptide specificity of c-Abl. Thus, these chimeric kinases are suitable for identifying the direct substrates of c-Abl. These engineered chimeric enzymes provide a new strategy for constructing kinases with tailor-made ligand binding properties.     

The complete amino acid sequence of the catalytic domain of rat brain hexokinase, deduced from the cloned cDNA

The complete amino acid sequence of the catalytic domain of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been deduced from the nucleotide sequence of cloned cDNA. Extensive similarity in sequence, taken to indicate similarity in secondary and tertiary structure, is seen between the mammalian enzyme and yeast hexokinase isozymes A and B. All residues critical for binding glucose to the yeast enzyme are conserved in brain hexokinase. A location for the substrate ATP binding site is proposed based on relation of structural features in the yeast enzyme to characteristics commonly observed in other nucleotide binding enzymes; sequences in regions proposed to be important for binding of ATP to the yeast enzyme are highly conserved in brain hexokinase.     

Substrate binding and reduction of benzoyl-CoA reductase: evidence for nucleotide-dependent conformational changes

Benzoyl-CoA reductase (BCR) from the denitrifying bacterium Thauera aromatica catalyzes the ATP driven two-electron reduction of the aromatic moiety of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene (2 ATP/2 e(-)). The enzyme contains two similar but nonidentical ATP-binding sites of the acetate kinase/sugar kinase/Hsp70/actin family. To obtain further insights into the overall catalytic cycle of BCR, the binding affinities and stoichiometries of all substrates as well as their effects on reduction kinetics were studied by stopped-flow UV/vis spectroscopy, freeze-quench EPR spectroscopy, and equilibrium dialysis. BCR bound maximally two nucleotides and a single BCoA. The binding of a single nucleotide induced a molecular switch (BCR --> BCR) as evidenced as follows: (i) the reduction rate of BCR by sulfoxide radical anion was significantly decreased in the nucleotide-bound state, (ii) the binding of BCoA to the reduced enzyme strictly depended on bound nucleotides, and (iii) the nucleotide binding affinities increased up to 60-fold compared to the steady-state values. The "ATP-binding switch" is distinguished from the previously described "low-spin/high-spin switch" of a [4Fe-4S] cluster which strictly depends on ATP hydrolysis. The two nucleotide binding sites were occupied sequentially; binding constants of the two sites differed by a factor of 10-40. The kinetic data obtained suggest that the ATP-binding switch is a rather fast process (>100 s(-)(1)) with a switch equilibrium constant of 54 +/- 10. In contrast, the reverse switch back of the MgADP-bound enzyme (BCR --> BCR) is considered rate-limiting in the overall catalytic cycle of BCR (4 +/- 1 s(-)(1)). The binding of nucleotides did not affect the redox potential of the [4Fe-4S](+1/+2) clusters; the switch is rather considered to alter the kinetics of internal electron transfer. Implications for the overall catalytic cycle of benzoyl-CoA reductase are discussed and compared with other ATP-hydrolyzing enzymes.     

The stem region of the sulfotransferase GlcNAc6ST-1 is a determinant of substrate specificity

The GlcNAc-6-sulfotransferases are a family of Golgi-resident enzymes that modulate glycan function. Two members of this family, GlcNAc6ST-1 and -2, collaborate in the biosynthesis of ligands for the leukocyte adhesion molecule L-selectin. Although their biochemical properties are similar in vitro, the enzymes have distinct glycoprotein substrate preferences in vivo. The sulfotransferases share similar overall architecture with the exception of an extended stem region in GlcNAc6ST-1 that is absent in GlcNAc6ST-2. In this study we probed the importance of the stem region with respect to substrate preference, localization, and oligomerization. Analysis of truncation mutants demonstrated that perturbation of the stem region of GlcNAc6ST-1 affects the cellular substrate preference of the enzyme without altering its retention within the Golgi. A chimeric enzyme comprising the stem region of GlcNAc6ST-1 inserted between the catalytic and transmembrane domains of GlcNAc6ST-2 had the same substrate preference as native GlcNAc6ST-1. In cells, GlcNAc6ST-1 exists as a dimer; two cysteine residues within the stem and transmembrane domain were found to be critical for dimerization. However, disruption of the dimer by mutagenesis did not affect either localization or substrate preference. Collectively, these results indicate that the stem region of GlcNAc6ST-1 influences substrate specificity, independent of its role in dimerization or Golgi retention.     

Nucleotide binding to the isolated beta subunit of the chloroplast ATP synthase.

The beta subunit isolated from the chloroplast ATP synthase F1 (CF1) has a single dissociable nucleotide binding site, consistent with the proposed function of this subunit in nucleotide binding and catalysis. The beta subunit bound the nucleotide analogs trinitrophenyl-ATP (TNP-ATP) or trinitrophenyl-ADP (TNP-ADP) with nearly equal affinities (Kd = 1-2 microM) but did not bind trinitrophenyl-AMP. Both ATP and ADP effectively competed with TNP-ATP for binding. Other nucleoside triphosphates were also able to compete with TNP-ATP for binding to beta; their order of effectiveness (ATP greater than GTP, ITP greater than CTP) mimicked the normal substrate specificity of CF1. The single nucleotide binding site on the isolated beta subunit very closely resembles the low affinity catalytic site (site 3) of CF1 (Bruist, M.F., and Hammes, G. G. (1981) Biochemistry 20, 6298-6305), suggesting that tight nucleotide binding by other sites on the enzyme involves other CF1 subunits in addition to the beta subunit. The results are inconsistent with an earlier report (Frasch, W.D., Green, J., Caguial, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069), which suggested more than one nucleotide binding site per beta subunit. Binding of nucleotides to the isolated beta subunit was eliminated by a brief heat treatment (40 degrees C for 10 min) of the protein. A small change in the circular dichroism spectrum of beta accompanied the heat treatment indicating that a localized (rather than global) change in the folding of beta, involving at least part of the nucleotide binding domain, had occurred. Also accompanying the loss of nucleotide binding was a loss of the reconstitutive capacity of the beta subunit. ATP protected against the effects of the heat treatment.     

pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains

Nucleotide binding to sarcoplasmic reticulum vesicles was investigated in the absence of calcium using both filtration and fluorescence measurements. Filtration assays of binding of radioactive nucleotides at concentrations up to 0.1 mM gave a stoichiometry of one ATP-binding site/sarcoplasmic reticulum ATPase molecule. When measured in the presence of calcium under otherwise similar conditions, ATPase velocity rose 4-8-fold (depending on pH and magnesium concentration) when the ATP concentration was increased from 1 microM to 0.1 mM. Binding of ATP and ADP enhanced the intrinsic fluorescence of sarcoplasmic reticulum ATPase, but AMP and adenosine did not affect it. Both filtration and fluorescence measurements showed that binding of metal-free ATP is independent of pH (Kd = 20-25 microM) but that the presence of magnesium induces pH dependence of the binding of the Mg.ATP complex (Kd = 10 microM at pH 6.0 and 1.5 microM at pH 8.0). Binding of metal-free ADP was pH-dependent but was not affected by magnesium. High magnesium concentrations inhibited nucleotide binding. These results suggest that ATP interacts with two different domains of Ca-ATPase that form the catalytic site. The first domain may bind the adenine moiety of the substrate, and the pH dependence of ADP binding suggests the participation of His683 in this region. The second domain of the catalytic site may bind the gamma-phosphate and the magnesium ion of the Mg.ATP complex and constitute the locus of the electrostatic interactions between the substrate and the enzyme.     

GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism

Sialic acid is a major determinant of carbohydrate-receptor interactions in many systems pertinent to human health and disease. N-Acetylmannosamine (ManNAc) is the first committed intermediate in the sialic acid biosynthetic pathway; thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. UDP-GlcNAc 2-epimerase and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has been shown to direct metabolic flux toward sialic acid in vivo, the function of the latter enzyme is unclear. Here we study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.     

Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.     

The human erythrocyte sugar transporter is also a nucleotide binding protein

We have previously shown that ATP interacts with an intracellular, stereoselective, regulatory site(s) on the human erythrocyte sugar transport system to modify transport function in a hydrolysis-independent manner. This present study examines the nucleotide binding properties of the human erythrocyte sugar transport system. We demonstrate by transport studies in ghosts, by nucleotide binding studies with purified transport protein by measurements of nucleotide inhibition of 8-azidoadenosine 5'-[gamma-32P]triphosphate (azido-ATP) photoincorporation into purified carrier, and by analysis of nucleotide inhibition of carboxyl-terminal peptide antisera binding to purified glucose carrier than the glucose transport protein binds (with increasing order of affinity) AMP, ADP, ATP, 5'-adenylyl imidodiphosphate (AMP-PNP), and 1,N6-ethenoadenosine 5'-triphosphate (EATP) at a single site. The carrier lacks detectable ATPase activity and GTP binding capacity. While AMP and ADP bind to the carrier protein and act as competitive inhibitors of ATP binding, these nucleotides are unable to mimic the ability of ATP, AMP-PNP, and EATP to modify the catalytic properties of the sugar transport system. Limited tryptic digestion of azido-ATP-photolabeled carrier suggests that the region of the glucose transport protein containing the intracellular cytochalasin B binding and extracellular bis(mannose) binding domains [residues 270-456; Holman, G. D., & Rees, W. D. (1987) Biochim. Biophys. Acta 897, 395-405] may also contain the intracellular ATP binding site.     

Tightly bound nucleotides affect phosphate binding to mitochondrial F1-ATPase

The interaction of inorganic phosphate with native and nucleotide-depleted F1-ATPase was studied. F1-ATPase depleted of tightly bound nucleotides loses the ability to bind inorganic phosphate. The addition of ATP, ADP, GTP and GDP but not AMP, restores the phosphate binding. The nucleotides affecting the phosphate binding to F1-ATPase are located at the catalytic (exchangeable) site of the enzyme. The phosphate is thought to bind to the same catalytic site where the nucleotide is already bound. It is thought that ADP is the first substrate to bind to F1-ATPase in the ATP synthesis reaction.     

Effect of ligand binding on the tryptic digestion pattern of rat brain hexokinase: relationship of ligand-induced conformational changes to catalytic and regulatory functions

The Type I isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is comprised of N- and C-terminal domains, associated with regulatory and catalytic functions, respectively. Extensive sequence similarity between the domains is consistent with evolution of the enzyme by gene duplication and fusion. Cleavage at tryptic sites located in the C-terminal domain is markedly sensitive to ligands present during digestion, while analogous sites in the N-terminal domain are either resistant to trypsin or unaffected by the presence of ligands. These results imply a lack of structural equivalence between the N- and C-terminal domains, with the overall structure of the N-terminal domain being "tighter" and with a major component of ligand-induced conformational changes being focused in the C-terminal domain. Based on a previously proposed structure for brain hexokinase, protection by substrate hexoses is attributed to substrate-induced closing of a cleft in the C-terminal domain. Similar protection at C-terminal cleavage sites results from binding of inhibitory hexose-6-phosphates to the N-terminal domain. In addition, hexose-6-phosphates evoke cleavage at a site, T5, located in a region that has been associated with binding of ATP to the C-terminal domain. Thus, alterations in this region, coupled with reduced accessibility resulting from cleft closure, may account for the mutually exclusive binding of inhibitory hexose-6-phosphates and substrate ATP. In the absence of Mg2+, all nucleoside triphosphates examined (ATP, UTP, CTP, and GTP) protected against digestion by trypsin. In contrast, ATP-Mg2+ stabilized the C-terminal domain but destabilized the N-terminal domain, while the chelated forms of the other nucleoside triphosphates were similar to the unchelated forms in their effect on proteolysis; the unique response to ATP-Mg2+ reflects the specificity for ATP as a substrate.