Apparent Affinity For ATP Research Articles

The effect of metal ions on the Spf1p P5A-ATPase. High sensitivity to irreversible inhibition by zinc

The Spf1p protein from Saccharomyces cerevisiae belongs to the family of P5A-ATPases that have recently been shown to protect the endoplasmic reticulum by dislocating misinserted membrane proteins. The loss of function of P5A-ATPases leads to endoplasmic reticulum stress with a pleiotropic phenotype including protein, sterol and metal ion dyshomeostasis. Like other P-ATPases, Spf1p requires Mg2+. We found that free Mg2+ stimulated the Spf1p ATPase activity along a double hyperbolic curve with two components of K1/2 = 14 and 800 μM Ca2+, Mn2+ and Co2+ lowered about 50% of the Spf1p ATPase with relatively low affinity (Ki ∼75 μM) and the activity was fully recovered after metal ion chelation with EGTA. In contrast, low concentrations of Zn2+ and Cd2+decreased the activity to less than 20% and lead to slow irreversible inactivation of the enzyme. After the treatment with Zn2+, Spf1p exhibited a reduced apparent affinity for ATP and formed lower levels of the catalytic phosphoenzyme. The inactivation by Zn2+ occurred preferentially at a pH > 6 and could be prevented by adding either ATP or ADP to the inactivation media. These results suggest that Zn2+ inactivated Spf1p by binding to amino acid residues from the nucleotide binding-phosphorylation domains that are protonated at lower pH. Alternatively the binding of nucleotides may indirectly compete with a conformational change leading to the Zn2+-inactive form of the enzyme. Exposure of yeast cells to high concentrations of Zn2+ led to changes similar to the phenotype characteristic of the Spf1Δ cells. Altogether, our data, point out a possible mechanism by which the inhibition of P5A-ATPases could potentiate metal ion–induced ER stress and proteotoxicity.

P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity.

Background and PurposeP2X receptors are trimeric ligand‐gated ion channels that open a cation‐selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains.Experimental ApproachHere, we examine the impact of P2X2 receptor inter‐subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches.Key ResultsWe demonstrate that predicted extracellular domain inter‐subunit interfaces display a higher‐than‐expected density of missense variations and that the majority of mutations that disrupt putative inter‐subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity.Conclusion and ImplicationsWe provide the first structural mapping of the mutational distribution across the human population in a ligand‐gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand‐gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

Lipid composition modulates ATP hydrolysis and calcium phosphate mineral propagation by TNAP-harboring proteoliposomes

Bone biomineralization is mediated by a special class of extracellular vesicles, named matrix vesicles (MVs), released by osteogenic cells. The MV membrane is enriched in sphingomyelin (SM), cholesterol (Chol) and tissue non-specific alkaline phosphatase (TNAP) compared with the parent cells’ plasma membrane. TNAP is an ATP phosphohydrolase bound to cell and MV membranes via a glycosylphosphatidylinositol (GPI) anchor. Previous studies have shown that the lipid microenvironment influences the catalytic activity of enzymes incorporated into lipid bilayers. However, there is a lack of information about how the lipid microenvironment controls the ability of MV membrane-bound enzymes to induce mineral precipitation. Herein, we used TNAP-harboring proteoliposomes made of either pure dimyristoylphosphatidylcholine (DMPC) or DMPC mixed with either Chol, SM or both of them as MV biomimetic systems to evaluate how the composition modulates the lipid microenvironment and, in turn, TNAP incorporation into the lipid bilayer by means of calorimetry. These results were correlated with the proteoliposomes’ catalytic activity and ability to induce the precipitation of amorphous calcium phosphate (ACP) in vitro. DMPC:SM proteoliposomes displayed the highest efficiency of mineral propagation, apparent affinity for ATP and substrate hydrolysis efficiency, which correlated with their highest degree of membrane organization (highest ΔH), among the tested proteoliposomes. Results obtained from turbidimetry and Fourier transformed infrared (FTIR) spectroscopy showed that the tested proteoliposomes induced ACP precipitation with the order DMPC:SM>DMPC:Chol:SM≈DMPC:Chol>DMPC which correlated with the lipid organization and the presence of SM in the proteoliposome membrane. Our study arises important insights regarding the physical properties and role of lipid organization in MV-mediated mineralization.

Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase

The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.

Structure/function studies of Diverse Forms of ADPGlucose Pyrophosphorylase

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that serves as the rate‐limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties in accord with adaptation to different environments; some of these properties would be useful to incorporate into crops to enhance starch production.The enzyme from Deinococcus radiodurans (D. rad), coded for by the glgC gene, is an extremophile resistant to radiation and harsh growth conditions. Using standard assays with magnesium (Mg) as a co‐factor, the purified D. rad enzyme was found to have relatively low apparent affinity for the substrate ATP and Mg with S0.5 values of 8.4 mM and 24 mM, respectively. However, this organism is known to have a high intracellular manganese (Mn) concentration, which correlates with ionizing radiation and desiccation resistance. Because stored glycogen could act as an energy and carbon source during cell recovery, it was hypothesized that this ADPG PPase may be more responsive to Mn. Use of Mn in the assay revealed a ~9.3 fold increase in apparent affinity for ATP, a ~5 fold increase in apparent affinity for Mn (versus Mg), and a two‐fold increase in Vmax which supports the hypothesis. Complete kinetic characterization of the wild‐type (WT) and selected altered enzymes is underway to probe the putative metal binding site(s). Several crystallization conditions were found to yield promising results and further crystallization trials are in progress.In contrast, the enzyme from the thermophile Thermotoga maritima (T.ma) consists of two subunits, coded for by the glgC and glgD genes. Previous studies indicated that the purified WT T.ma glgD alone had no activity but was found to stimulate the relatively low activity of the glgC subunit. The complex could also be activated by FBP while the glgC alone was insensitive to FBP. The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. Previous results indicated that limited proteolysis of the C‐terminus of glgD resulted in lower activation of the complex. In order to probe this further, a 73 amino acid C‐terminal truncation was generated and the activity of the complexes with the WT and WT glgC/truncated glgD (altered complex) compared. The altered complex exhibited a lower apparent affinity for ATP than the WT complex as well as a 5‐fold reduction in Vmax, which still represented a ~3‐fold stimulation compared to the glgC alone. In the presence of the activator FBP, the altered complex was slightly inhibited by FBP in contrast to the 2‐fold activation of the WT complex. The results appear to support a role for the C‐terminus in allosteric activation by FBP and partial stimulation of the enzyme in the absence of FBP. A 3 Å crystal structure for the glgC subunit was recently solved which was in good agreement with a previously modeled structure based on the A. tumefaciens enzyme structure (PDB 3BRK). Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of various complexes are underway.Support or Funding InformationSupported in part by NSF BIO MCB Grant 0448676 and BIO DBI.

Role of the inter‐subunit surface interaction in the regulation of the ADP‐glucose pyrophosphorylase from Agrobacterium tumefaciens

ADP‐glucose pyrophosphorylase (ADP‐Glc PPase) is a key regulatory enzyme involves in the starch and glycogen synthesis for plants and bacteria respectively, which catalyzes the synthesis of ADP‐glucose from glucose 1‐phosphate (Glc1P) and ATP. ADP‐Glc PPase from Agrobacterium tumefaciens is allosterically regulated by fructose 6‐phosphate (Fru6P) and pyruvate (Pyr). Previously, it was observed that removal of a disulfide bridge between the α‐subunits of the potato tuber enzyme resulted in a more active enzyme [1], indicating that inter‐subunit interactions are important in the allosteric mechanism [2]. By studying the structure of the ADP‐Glc PPase enzyme of Agrobacterium tumefaciens, it comes to light that D141 is interacting with the R11 of the opposing subunit [3], which was suggested to be involved in activation by pyruvate [4]. To prove our hypothesis that D141 and R11 residues are involved in regulation, we made three single mutants: D141A, D141R, R11D, and the double mutant R11D‐D141R. D141A exhibited reduced Vmax values in the absence of activators (~4‐fold), reduced apparent affinity for Fru6P (~2.5‐fold), and lower apparent affinity for ATP (~3‐fold). In the presence of Fru6P, D141A exhibited only ~4.3% of the wild‐type activity, and affinity for ATP was not affected as well indicating a virtual insensitivity to Fru6P. When D141R was compared to wild‐type enzyme, the activity lowered ~3600‐fold. For D141R the apparent affinity for ATP was decreased in the presence of Fru6P. R11D was similarly active as the wild‐type, while it is completely insensitive to both activators Fru6P and Pyr. The double mutant R11D‐D141R was showing decreased activity (~95‐fold) in compared to wild type, and insensitive to both activators. Our results suggest that the functional role of the D141 and R11 residues is to interacting with the opposing subunit and enabling the regulation of the ADP‐Glc PPase.Support or Funding InformationNational Science Foundation

Structure‐Function Studies of Deinococcus radiodurans ADP Glucose Pyrophosphorylase: Role of Ser48 in Allosteric Regulation

Adenosine Diphosphate Glucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that functions as the rate‐limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield in crops. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties; some of these properties would be useful to incorporate into transgenic crops to enhance starch production. While a number of ADPG PPases have been kinetically characterized, there is only one published x ray structure (PDB 3BRK) of an inhibited form. Very little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile that is resistant to ionizing radiation and harsh growth conditions. When comparing the amino acid sequence of this enzyme to other characterized ADPG PPases, it was noted that position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of Ser‐48, the S48A enzyme was generated by site‐directed mutagenesis and the recombinant altered D. rad ADPG PPases successfully expressed in E. coli and purified via a scheme that includes anion exchange chromatography, size exclusion chromatography, and affinity chromatography. Initial studies on the S48A enzyme in the absence of activators have shown a dramatic 20‐fold increase in the apparent affinity for the substrate ATP, a 3‐fold increase in apparent affinity for the cofactor magnesium, and a 4‐fold increase in Vmax compared to wild‐type. Interestingly, in the presence of the activator FBP there was little change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 14‐fold increase in Vmax and 12‐fold and 3‐fold increase in apparent affinity for ATP and magnesium, respectively. Similarly, in the presence of F6P, there were not a very large increase in Vmax or higher apparent affinity for ATP for the S48A enzyme, but there was a 9‐fold difference in Vmax and a 12‐fold and 2‐fold increase in apparent binding affinity for ATP and magnesium for WT, respectively. The WT enzyme also displayed higher apparent affinity for FBP and F6P compared to the S48A enzyme. The alanine substitution appears to result in an enzyme form that is partially activated but relatively insensitive to activators. Further kinetic characterization of S48A in the presence of sulfate and the inhibitor phosphate is in progress. With respect to physical characterization, two crystallization conditions for S48A, one including imidazole and the other with lithium sulfate and PEG, were found to yield preliminary results. Further crystallization trials are in progress as a first step in elucidating the three‐dimensional structure.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Structure‐function studies of ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii: Probing the Role of the C‐terminus in Allosteric Regulation

Starch serves as both a critical food source and an important biodegradable and renewable carbon source for biofuel and other industries including adhesives, packaging material, and cosmetics. ADP‐glucose pyrophosphorylase (ADPG PPase, glgC gene product) is the key enzyme that is responsible for the rate‐limiting step of both glycogen and starch biosynthesis in bacteria and plants, catalyzing the conversion of glucose‐1‐phosphate and ATP to ADPG and pyrophosphate. Thermodesulfovibrio yellowstonii (Td. y) is a thermophilic sulfate‐reducing bacterium that was isolated from geothermal vents in Yellowstone National Park. Little is known about this ADPG PPase which displays increased heat stability, salt tolerance, and altered regulation compared to other ADPG PPase family members. The Td.y enzyme is substantially activated by phosphoenolpyruvate (PEP), which differs from many other ADPG PPases, as well as G6P, 3PGA, and sulfate. These properties could contribute to biomass production in different crop environments. Multiple sequence alignments have shown deviations from consensus residues in the N‐terminal allosteric region and C‐terminal region of the Td.y ADPG PPase. Two of the more interesting differences are at positions 365 and 397, which have been shown to be involved in allosteric regulation of other ADPG PPases, as part of a conserved region in the enzyme family and associated with plant enzymes, respectively. The K365G and R397A enzymes were generated by site‐directed mutagenesis as a first step in probing structure‐function relationships. We hypothesize that the positively charged lysine at 365 and arginine at 397 may contribute to PEP binding and activation. The altered proteins were purified to homogeneity by a procedure that included two hydroxyapatite chromatography steps followed by size exclusion chromatography. Initial kinetic studies revealed that the K365G protein displayed a lower Vmax (~25% of wild‐type [WT]) but ~2‐fold higher apparent affinity for ATP in the absence of effectors. In contrast to the WT, which was activated over 3‐fold by PEP, the K365G enzyme only exhibited ~10% activation by PEP and a decrease in apparent affinity for ATP. Conversely, the K365G enzyme was activated 2.6 fold by sulfate, an effector which has a much smaller effect on the Vmax of WT. In the absence of effectors, the R397A displayed a similar apparent affinity for ATP compared to WT but a 24 fold decrease in Vmax. In the presence of PEP, R397A exhibited lower apparent affinity for ATP and a Vmax decreased ~42 fold. The response of this enzyme to G6P was very similar to WT. The results so far support the hypothesis that K365 and R397 are critical for PEP activation while also influencing other allosteric properties. Complete kinetic analyses of the K365G and R397A enzymes as well as crystallization trials are underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Structure‐Function Studies of the Thermotoga maritima ADPGlucose Pyrophosphorylase: Probing the role of the C‐terminus of the GlgD subunit

ADP Glucose pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step of glycogen and starch biosynthesis in plants and bacteria. Structure‐function studies of the enzyme from different sources will allow the engineering of a more active enzyme to produce more renewable and biodegradable carbon. In Thermotoga maritima (T.ma) there are two genes, glgC and glgD, which code for two subunits of this ADPG PPase. The recombinant His‐tagged subunits were successfully purified using a heat step and nickel chromatography. Previous studies performed at 37ºC (pH 7.5) had shown that the wild type (WT) glgD alone had no activity, while the glgC protein displayed a specific activity of ~ 3 Unit/mg with S0.5 values for ATP and magnesium (Mg2+) of 5.39 mM and 18.66 mM, respectively. However, the WT glgC/D complex at subsaturating substrate conditions was stimulated ~20‐fold compared to glgC alone and was also activated ~2‐fold by fructose‐1,6‐ bisphosphate (FBP). The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. In the presence of FBP, the apparent affinity for ATP increased ~6‐fold for the complex with a modest ~30% increase in Vmax. Previous alignment studies and molecular modeling principally focused on the functional role of the N‐terminus. Therefore the truncation of 73 amino acids at the C‐terminal end of glgD was generated to test its potential functional role in the complex. In the absence of FBP, the truncated complex exhibited a modest decrease in ATP apparent affinity for ATP and Mg2+ with ~20‐fold and ~2 fold decreased in Vmax, respectively, compared to the WT complex. It was noted that in the presence of the truncated glgD, the Vmax was substantially lower than for glgC alone, indicating an inhibitory role. In the presence of FBP, the truncated complex displayed a ~6‐fold decrease in apparent affinity for ATP, ~3‐fold for Mg and a ~6‐fold lower Vmax compared to the WT complex. However, the Vmax of the truncated complex is close to the Vmax of the glgC alone indicating that FBP diminishes the inhibition by the truncated glgD. The primary effect of the truncation appears to be on the Vmax of the complex as well as a reduction in the K‐type activation by FBP observed with the WT. Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of WT GlgC/WT GlgD are still underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Probing the role of the N‐terminus in the ADP‐Glucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii

Glucans serve as critical energy storage compounds in nearly all living organisms. Such renewable and biodegradable carbon sources are in demand now from industry as an eco‐friendly substitute for petroleum based products. ADP‐Glucose Pyrophosphorylase (ADPG PPase) is the enzyme that catalyzes the rate limiting step of glucan biosynthesis in plants and bacteria, respectively. The novel Thermodesulfovibrio yellowstonii (Td.y) ADPG PPase, from a thermophilic, sulfate‐reducing bacterium, has potential properties that could lead to engineering a very stable and highly active form which could increase the yield of starch. Further, sequence alignment data indicate that Td.y harbors varied amino acids in two conserved N‐terminal regions (glycine rich and RRAKPAV) that are known to be important for regulation and catalysis in other characterized ADPG PPases. The recombinant Wild‐type (WT) and variants (E15A, F23R, S25A) have been successfully purified by hydroxyapatite and size exclusion chromatography. In the case of E15A, preliminary data displayed similar velocity and similar apparent substrate affinity for ATP compared to WT, in the absence of effector molecules. However, in the presence of the activator PEP, E15A shows a ~7 fold increase in activity compared to 4 fold in WT. This suggests a role in regulation and binding of effector molecules. In the absence of effector molecules, preliminary data from F23R reveal a 4 fold decrease in activity and a ~2 fold decrease in apparent affinity for ATP compared to the WT. However, in the presence of effector molecules, ATP affinity was restored to WT values. In the presence of PEP and FBP, F23R displays a ~7 fold increase in activation compared to WT. The F23 position appears to play a role in structure or folding/allosteric regulation, which is also supported by molecular modeling. These results suggest a potential for altered binding of negatively charged metabolites due to increased positively charged residues in the postulated binding pocket. Kinetic data for the variant S25A reveals a Vmax ~3 fold higher than WT with a10‐fold higher apparent affinity for ATP compared to the WT as well as desensitization to effector molecules. This position in Td.y may play a role in setting the relative activity level that can be further attenuated by effectors. Along with kinetic analyses, initial crystallization trials are underway for variant S25A with initial crystals from different conditions diffracting to a resolution of ~3.96 Å. Results gathered from the WT and variants thus far confirm and extend the role of the two conserved regions for activity and regulation.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator.

Two functional abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR), a 25% reduction of the single-channel conductance (g) and a ∼13-fold lower open probability (Po ), were found with the R117H mutation that is associated with mild forms of cystic fibrosis. Characterizations of the gating defects of R117H-CFTR led to the conclusion that the mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains (TMDs) without altering the function of the nucleotide binding domains (NBDs). Nonetheless, gating of the R117H-CFTR can be improved by a variety of pharmacological reagents supposedly acting on NBDs such as ATP analogues, or TMDs (e.g. VX-770 or nitrate). These reagents potentiate synergistically R117H-CFTR gating to a level that allows accurate assessments of its gating deficits. Our studies not only elucidate the mechanism underpinning gating dysfunction of R117H-CFTR, but also provide a mechanistic understanding of how VX-770 ameliorates the gating defects in the R117H mutant. Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated, but ATP-gated chloride channel. In the current study, we investigated the mechanism responsible for the gating defects manifested in R117H-CFTR, an arginine-to-histidine substitution at position 117 of CFTR that is associated with mild forms of CF. We confirmed previous findings of a 25% decrease of the single-channel conductance (g) in R117H-CFTR, but found a ∼13-fold lower open probability (Po ). This dramatic gating deficit is not due to dysfunctional nucleotide binding domains (NBDs) as the mutation does not alter the apparent affinity for ATP, and the mutant channels respond to ATP analogues in a similar manner as wild-type CFTR. Furthermore, once ATP hydrolysis is abolished, the R117H mutant can be trapped in a prolonged 'burst opening' conformation that is proposed to be equipped with a stable NBD dimer. On the other hand, our results support the conclusion that the R117H mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains as even when NBDs are kept at a dimerized configuration, Po is reduced by ∼10-fold. Moreover, our data demonstrate that a synergistic improvement of R117H-CFTR function can be accomplished with a combined regiment of VX-770 (Ivacaftor), nitrate ion (NO3 (-) ) and N(6) -(2-phenylethyl)-2'-deoxy-ATP (d-PATP), which almost completely rectifies the gating defect of R117H-CFTR. Clinical implications of our results are discussed.

Probing the Role of Ser‐48 in the Regulation of Deinococcus radiodurans ADP Glucose Pyrophosphorylase

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that serves as the rate-limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties; some of these properties would be useful to incorporate into crops to enhance starch production. Little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile resistant to radiation and harsh growth conditions. When comparing the sequence of this enzyme to others, position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of S48, the S48A enzyme was generated by site-directed mutagenesis and the altered D. rad ADPG PPase successfully expressed and purified. Initial studies on the S48A enzyme in the absence of activators have shown a 20-fold increase in the apparent affinity for the substrate ATP and a 4-fold increase in Vmax compared to wild-type. Interestingly, in the presence of the activators FBP and F6P there were no change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 5-fold increase in Vmax and 12-fold and 3-fold increase in apparent affinity for ATP and magnesium, respectively. The alanine substitution appears to result in an enzyme form that is partially activated but insensitive to activators. Complete kinetic analyses of the S48A enzyme are in progress. Supported in part by NSF Grant 0448676.

Correction to The Specialized Hsp70 (HscA) Interdomain Linker Binds to Its Nucleotide-Binding Domain and Stimulates ATP Hydrolysis in Both cis and trans Configurations

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron-sulfur (Fe-S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min(-1) at 25 °C, henceforth reported in units of min(-1)). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA's interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min(-1)) is nearly 15-fold higher than that of HscA386 (0.035 min(-1)), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower (1)H(N) line widths in its two-dimensional (1)H-(15)N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L(387)LLDVIPLS(395)) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.

A kinetic characterization of the gill V(H(+))-ATPase in juvenile and adult Macrobrachium amazonicum, a diadromous palaemonid shrimp.

Novel kinetic properties of a microsomal gill V(H(+))-ATPase from juvenile and adult Amazon River shrimp, Macrobrachium amazonicum, are described. While protein expression patterns are markedly different, Western blot analysis reveals a sole immunoreactive band, suggesting a single V(H(+))-ATPase subunit isoform, distributed in membrane fractions of similar density in both ontogenetic stages. Immunofluorescence labeling locates the V(H(+))-ATPase in the apical regions of the lamellar pillar cells in both stages in which mRNA expression of the V(H(+))-ATPase B-subunit is identical. Juvenile (36.6±3.3 nmol Pi min(-1) mg(-1)) and adult (41.6±1.3 nmol Pi min(-1) mg(-1)) V(H(+))-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K0.5=0.21±0.02 mmol L(-1)) being 3-fold greater than for juveniles (K0.5=0.61±0.01 mmol L(-1)). The K0.5 for Mg(2+) interaction with the juvenile V(H(+))-ATPase (1.40 ± 0.07 mmol L(-1)) is ≈6-fold greater than for adults (0.26±0.02 mmol L(-1)) while the bafilomycin A1 inhibition constant (KI) is 45.0±2.3 nmol L(-1) and 24.2±1.2 nmol L(-1), for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of ≈25 nmol Pi min(-1) mg(-1), suggesting the presence of ATPases other than the V(H(+))-ATPase. These differences may reflect a long-term regulatory mechanism of V(H(+))-ATPase activity, and suggest stage-specific enzyme modulation. This is the first kinetic analysis of V(H(+))-ATPase activity in different ontogenetic stages of a freshwater shrimp and allows better comprehension of the biochemical adaptations underpinning the establishment of palaemonid shrimps in fresh water.

The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron–sulfur (Fe–S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min−1 at 25 °C, henceforth reported in units of min–1). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min–1) is nearly 15-fold higher than that of HscA386 (0.035 min–1), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower 1HN line widths in its two-dimensional 1H–15N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L387LLDVIPLS395) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.

Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm(-1) s(-1), followed by rate-limiting ADP release at 12.8 s(-1). ATP binding at 7.5 μm(-1) s(-1) was followed by an ATP-promoted isomerization at 84 s(-1) to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s(-1). ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s(-1). The dissociation step showed an apparent affinity for ATP that was very weak (K½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm(-1) s(-1), which is inconsistent with fast ATP binding at 7.5 μm(-1) s(-1) and a Kd ,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K½,ATP at 133 μm. The steady-state ATPase Km ,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.

The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor

P2X7, an ATP-gated cation channel, is involved in immune cell activation, hyperalgesia and neuropathic pain. By regulating cytokine release in the brain, P2X7 has been linked to the pathophysiology of mood disorders and schizophrenia. We here assess the impact of 123 drugs that act in the central nervous system on human P2X7. Most prominently, the tricyclic antipsychotics prochlorperazine (PCP) and trifluoperazine (TFP) potently inhibited P2X7-mediated Ca2+ entry, dye permeation and ionic currents. In divalent cation-containing bath solutions or after prolonged incubation, ATP-evoked P2X7 currents were inhibited by 10 μM PCP. This effect was not related to dopamine receptor antagonism. Surprisingly, PCP co-applied with ATP enhanced inward currents in bath solutions with low divalent cation concentrations. Intracellular perfusion with PCP did not substitute for the extracellularly applied drug, indicating that its binding sites are accessible from the extracellular space. Since P2X7 current potentiation by PCP was voltage-dependent, at least one site may be located within the electrical field of the membrane. While the channel opening and closure kinetic was altered by PCP, the apparent affinity of ATP remained unchanged (potentiation) or changed slightly (inhibition). Measurements in human monocyte-derived macrophages confirmed the PCP-induced inhibition of ATP-evoked Ca2+ influx, Yo-Pro-1 permeability, and whole cell currents. Interestingly, neither heterologously expressed rat or mouse P2X7 nor native P2X7 in rat astrocyte cultures or in mouse bone marrow-derived macrophages were inhibited by perazines with a similar potency. We conclude that perazine-type neuroleptics are potent, but species-selective allosteric modulators of human but not murine P2X7 receptors.

Structure‐Function Studies of the ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii

ADPGlucose pyrophosphorylase (ADPG PPase, glgC gene product) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. Engineering of this enzyme family will allow for the increased production of renewable carbon. The Thermodesulfovibrio yellostonii (Td.y) glgC gene has been successfully cloned and the recombinant enzyme purified. This enzyme displays only ~30% identity to other characterized ADPG PPases and harbors unusual sequences in regions involved in regulation. Molecular modeling studies revealed that the Td.y enzyme was most similar to a plant enzyme, in accord with 3‐phosphoglycerate (3‐PGA) activation. Heat stability up to 75°C required the presence of ATP. Initial kinetic studies were performed at 37°C (pH 7.5) and revealed S0.5 values for ATP and Mg of 8.0 mM and 13.9 mM, respectively, and a Vmax value of 5.33 Units/mg. The metabolites PEP (2mM), glucose‐6‐phosphate ( 2 mM), and 3‐PGA (2 mM), were found to increase the apparent affinity for ATP by 4.2, 10.3, and 3.8‐fold, respectively. PEP also increased the Vmax by 2.0‐fold. Based on alignment studies and molecular modeling, the following site‐directed mutants have been generated to probe regulatory properties: E15S, F18K, F23R, S25A, and S28A. Complete characterization of the native and altered proteins is underway. Supported by NSF Award 0448676 and a CSUPERB Presidents’ Commission Scholars grant to E. Yik and C. R. Meyer.

The Importance of a Loop Structure in ATP Apparent Affinity and Allosteric Activation of Escherichia coli ADP‐Glucose Pyrophosphorylase

Previous work into the study Escherichia coli ADP‐Glc PPase has identified several regions of the N‐terminal domain that appear to play a key role in the allosteric activation of the enzyme by Fructose‐1,6‐bisphosphate (FBP). One such region involves a loop structure formed by residues Pro103‐Gln116. Alanine scanning mutagenesis was performed on residues of this loop. The results of kinetic assays monitoring the forward (ADP‐Glc synthesis) reaction show that several residues play either a direct or indirect role in ATP binding or activation of the enzyme by FBP. These are Pro103, Gln105, Gln106, Arg107, Trp113, Tyr114, and Arg115. Aside from Arg115, whose main effect appears to have more to do with ATP binding than FBP activation, the defining characteristic of the alanine mutants was the failure to be appreciably activated by Fru‐1,6‐BP. These mutants saw near wild type activity in the absence of FBP but were only activated 1.5‐ to 5‐fold by FBP compared to 30‐fold for the wild type. Using a homology model of the E. coli enzyme (based on the crystal structures of closely related ADP‐Glc PPases from Agrobacterium tumefaciens and Potato tuber), we hypothesize some structure‐function relationships. For instance, the importance Gln105 and Gln106can likely be associated with hydrogen bond interactions with another loop structure formed by Tyr75‐Gln78 which has previously been identified as being important for ATP binding. We conclude that most of the loop is important to trigger the activation by FBP and increase the apparent affinity for ATP. Supported by funds from NSF MCB‐1024945

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K₀.₅ = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄⁺ had no modulatory effect. The affinity for Na⁺ (K₀.₅ = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.