Affinity For ADP Research Articles

Modulation of ATPase Activity by Physical Disengagement of the ATP-binding Domains of an ABC Transporter, the Histidine Permease

The membrane-bound complex of the prokaryotic histidine permease, a periplasmic protein-dependent ABC transporter, is composed of two hydrophobic subunits, HisQ and HisM, and two identical ATP-binding subunits, HisP, and is energized by ATP hydrolysis. The soluble periplasmic binding protein, HisJ, creates a signal that induces ATP hydrolysis by HisP. The crystal structure of HisP has been resolved and shown to have an "L" shape, with one of its arms (arm I) being involved in ATP binding and the other one (arm II) being proposed to interact with the hydrophobic subunits (Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., and Kim, S.-H. (1998) Nature 396, 703-707). Here we study the basis for the defect of several HisP mutants that have an altered signaling pathway and hydrolyze ATP constitutively. We use biochemical approaches to show that they produce a loosely assembled membrane complex, in which the mutant HisP subunits are disengaged from HisQ and HisM, suggesting that the residues involved are important in the interaction between HisP and the hydrophobic subunits. In addition, the mutant HisPs are shown to have lower affinity for ADP and to display no cooperativity for ATP. All of the residues affected in these HisP mutants are located in arm II of the crystal structure of HisP, thus supporting the proposed function of arm II of HisP as interacting with HisQ and HisM. A revised model involving a cycle of disengagement and reengagement of HisP is proposed as a general mechanism of action for ABC transporters.

Effects of Phosphorylation of Threonine 160 on Cyclin-dependent Kinase 2 Structure and Activity

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.

Sugar phosphorylation modulates ADP inhibition of maize mitochondrial hexokinase

The preference of maize (Zea mays L.) mitochondrial hexokinase (EC 2.7.1.1.) for glucose and fructose and the ADP regulation were evaluated. The Michaelis‐Menten constants (Km) varied between 0.02 and 0.09 mM for glucose and from 2 to 6 mM for fructose as substrates. The value of Vmax was five times higher in the presence of glucose as compared with fructose in membrane‐bound enzyme preparations. It was shown that ADP produced from the reaction inhibits the hexokinase activity (Ki=20–50 μM). However, the inhibition was very specific for adenine nucleotide. Only a small inhibition was observed when 1 mM of UDP, CDP or GDP was included in the assay medium. Nevertheless, the ADP inhibition was observed only when glucose was phosphorylated. In assay conditions where fructose serves as substrate, the affinity for ADP decreased by 10‐fold (Ki varied between 500 and 1 000 μM). These kinetics properties were also observed in partially purified soluble enzyme preparations. These data suggest that the type of hexose bound to the catalytic site modulates the ADP control of maize mitochondrial hexokinase.

Characterization of the nucleotide binding properties of SV40 T antigen using fluorescent 3'(2')-O-(2,4,6-trinitrophenyl)adenine nucleotide analogues.

ATP binding to the large tumor (T) antigen encoded by the simian virus 40 (SV40) genome plays an essential role in the replication of viral DNA [Fanning, E., and Knippers, R. (1992) Annu. Rev. Biochem. 61, 55-85]. To better explore the functions of T antigen during the replication process, we have studied the interactions of T antigen with fluorescent 3'(2')-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues. Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluorescence enhancement and a concomitant blue shift (11 nm) of the maximal emission wavelength; the intrinsic protein tryptophan fluorescence was quenched maximally by 50%. Both signals were utilized to characterize the nucleotide binding activity of T antigen. TNP-ATP and TNP-ADP bound to the ATP binding site with dissociation constants of 0.35 microM and 2.6 microM. TNP substitution enhanced the affinity of ADP for T antigen by approximately 11-fold. The binding stoichiometry was 1 mol of TNP nucleotide per mole of monomer T antigen. The binding of TNP-ATP was more temperature dependent than that of TNP-ADP. The enthalpy change contributed nearly half of the energy for TNP-ATP binding, whereas binding of TNP-ADP was primarily entropy driven. Both TNP-ATP and TNP-ADP were strong inhibitors of the T antigen ATPase activity, confirming the high affinities of the TNP nucleotides for the ATP binding site. Like the parent nucleotides, they also induced T antigen hexamer formation. Using the TNP nucleotides as fluorescent probes, we have measured the affinity of various nucleotides and analogues for T antigen. The results indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggesting closely related ATP binding sites in the three DNA helicases.

TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli.

The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli. This interaction results in autophosphorylation of CheA, a histidine protein kinase. Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors. As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals. To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides. Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently. Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP. Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP. Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner. TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM). The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made. Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase. This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA. These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA.

Site-directed Mutational Analysis for the ATP Binding of DnaA Protein: FUNCTIONS OF TWO CONSERVED AMINO ACIDS (LYS-178 AND ASP-235) LOCATED IN THE ATP-BINDING DOMAIN OF DnaA PROTEIN IN VITRO AND IN VIVO

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.

Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli.

The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400-450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 degreesC, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 degreesC resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.

Mitochondrial oxidative function in human saponin-skinned muscle fibres: effects of prolonged exercise.

1. The influence of prolonged exhaustive exercise on mitochondrial oxidative function was investigated in ten men. 2. Muscle biopsies were taken before and after exercise and mitochondrial respiration investigated in fibre bundles made permeable by pretreatment with saponin. 3. After exercise, respiration in the absence of ADP increased by 18 % (P < 0.01), but respiration at suboptimal ADP concentration (0.1 mM) and maximal ADP-stimulated respiration (1 mM ADP) remained unchanged. 4. In the presence of creatine (20 mM), mitochondrial affinity for ADP increased markedly and respiration at suboptimal ADP concentration (0.1 mM) was similar (pre-exercise) or higher (post-exercise; P < 0.05) than with 1 mM ADP alone. The increase in respiratory rate with creatine was correlated to the relative type I fibre area (r = 0.84). Creatine-stimulated respiration increased after prolonged exercise (P < 0.01). 5. The respiratory control index (6.8 +/- 0.4, mean +/- s.e.m.) and the ratio between respiration at 0.1 and 1 mM ADP (ADP sensitivity index, 0.63 +/- 0.03) were not changed after exercise. The sensitivity index was negatively correlated to the relative type I fibre area (r = -0.86). 6. The influence of exercise on muscle oxidative function has for the first time been investigated with the skinned-fibre technique. It is concluded that maximal mitochondrial oxidative power is intact or improved after prolonged exercise, while uncoupled respiration is increased. The latter finding may contribute to the elevated post-exercise oxygen consumption. The finding that the sensitivity of mitochondrial respiration for ADP and creatine are related to fibre-type composition indicates intrinsic differences in the control of mitochondrial respiration between fibres.

The Negative Dominant Effects of T340M Mutation on Mammalian Pyruvate Kinase

A fundamental issue in allosteric regulatory enzymes is the identification of pathways of signal transmission. Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address this issue because these isozymes exhibit different enzymatic regulatory patterns, and the sequence differences between these isozymes have identified the amino acid residues that alter their kinetic behavior. In an earlier study, Cheng et al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Chem. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the same mutation was effected in the kidney isozyme. Qualitatively, the same negative effects are observed in both isozymes, namely a significant decrease in catalytic efficiency and decrease in apparent affinity for phosphoenolpyruvate but no change in affinity for ADP, and a decrease in responsiveness to the presence of effectors, be it activator or inhibitor. Because the diversity in the primary sequence between these two isozymes does not alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative effect. The negative effects of T340M mutation on the kinetic properties imply that there is communication between residue 340 and the active site. Residue 340 is located at the 1,4 subunit interface; however, a T340M mutation enhances the dimerization affinity along the 1,2 subunit interface. Thus, this study has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.

Purification and Characterization of Hamster Liver Microsomal 7α-Hydroxycholesterol Dehydrogenase: SIMILARITY TO TYPE I 11β-HYDROXYSTEROID DEHYDROGENASE

While studying the bile acid synthetic pathway of hamsters, we discovered an NADP+-dependent liver microsomal 7alpha-hydroxycholesterol dehydrogenase (7alpha-HCD) activity that was not observed in rat liver microsomal fractions. The hamster liver microsomal 7alpha-HCD was purified to homogeneity using 2', 5'-ADP and cholic acid-agarose affinity chromatography. 7alpha-HCD displayed a molecular weight of approximately 34,000 on SDS-polyacrylamide gel electrophoresis; it is an intrinsic membrane protein of the hamster liver endoplasmic reticulum and exists as a multimeric aggregate in pure form. Partial N-terminal amino acid sequence analysis showed that 7alpha-HCD had high sequence similarity to human 11beta-hydroxysteroid dehydrogenase (11beta-HSD; 24/30 amino acid identity). The Km values for corticosterone and 7alpha-hydroxycholesterol were 1.2 and 1.9 microM, respectively, for purified 7alpha-HCD; both reactions displayed identical Vmax values (approximately 170 nmol/min/mg of protein). The IC50 of carbenoxolone, a competitive inhibitor of 11beta-HSD, was 75 nM for 7alpha-hydroxycholesterol dehydrogenation and 210 nM for corticosterone dehydrogenation. The tissue-specific expression in hamster was as follows: adrenal >/= liver > kidney > testis >> brain > lung. Microsomal 7alpha-HCD is uniquely expressed in hamster liver and to some extent in human liver but not in rat liver. Western blot analysis with two antibodies elicited against an N-terminal peptide of the human 11beta-HSD and purified hamster liver 7alpha-HCD, respectively, suggested the presence of multiple forms of 7alpha-HCD in hamster liver, most likely due to the existence of a family of 11beta-HSD proteins. Since 7-oxocholesterol is a potent inhibitor of cholesterol 7alpha-hydroxylase, alternative mechanisms for regulation of bile acid synthesis may exist in human and hamster liver due to production of this metabolite and its potential as an oxysterol.

Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms.

The striated muscle myosin of Placopecten moves actin faster in in vitro motility assays and has a higher actin-activated ATPase turnover rate than the myosin of the catch muscle. The heavy chain sequences of the two PlacoS1s are almost identical except at the surface loop 1 near the nucleotide binding pocket, where the two sequences vary significantly. Argopecten striated muscle myosin is 96% identical to Placopecten striated myosin, and both move actin with a similar velocity. To identify the individual kinetic steps which differ between these myosins, we completed a transient kinetic characterization of the three myosin S1s. The two striated S1s have similar rates of nucleotide binding to S1 and to acto.S1. The largest differences between the two are in the rate of ADP dissociation from S1 and affinity of ADP to S1, which differ by a factor of 2. The rates of nucleotide binding, nucleotide dissociation and affinity to nucleotides of the two Placopecten S1s are similar and agree within a factor of 2. In contrast, the affinity of acto.S1 for ADP is nine times weaker for the striated acto.S1 than for the catch acto.S1, compatible with the differences in motility of the Placopectenmyosins. Thus the differences in ADP affinity to acto.S1 and in the in vitro motility can be attributed to the differences in surface loop 1.

The M4M5 Cytoplasmic Loop of the Na,K-ATPase, Overexpressed in Escherichia coli, Binds Nucleoside Triphosphates with the Same Selectivity as the Intact Native Protein

Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase. A 1260-base DNA segment encoding Lys354-Lys774 of the rat alpha1-subunit was constructed via polymerase chain reaction. The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat alpha-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance. Bacterial cultures were grown, and protein synthesis was induced with isopropyl beta-D-thiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein ( approximately 46 kDa). The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded approximately 20 mg of soluble His6-alpha-loop/L cell culture. The His6-alpha-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid. More specifically, circular dichroism spectroscopy was used to estimate the secondary structure of the His6 loop, revealing an ordered folding composed of 23% alpha-helix, 23% antiparallel beta-sheet, 4% parallel beta-sheet, 19% beta-turn, and 32% random coil. The 6-histidine loop bound the fluorescent ATP analog trinitrophenyl-ATP with high affinity, as determined by measuring the fluorescence changes associated with binding. Affinities for ATP ( approximately 350 microM) and ADP ( approximately 550 microM) were determined by their ability to compete with and displace 2',3'-O-[2,4,6,-trinitrophenyl]-ATP. These nucleotide affinities are similar to those observed for the E2 conformation of the intact Na,K-ATPase.

The use of fast kinetic systems for the study of ng-microgram quantities of purified muscle proteins.

Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However these methods traditionally require relatively large amounts of protein (several mg) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g. Dictyostelium). We have been developing methods which would allow fast transients to be studied on the much smaller quantities of protein available from individual drosophila muscles, single mammalian muscle fibres, human biopsies and non-muscle myosins. The use of fluorescent labels on actin, myosin or ATP can be used in modern stopped-flow equipment at concentrations as low as 10 nM. These labels can report on the interactions between the three molecules using a few microgram of protein. However the stopped flow systems requires large volumes of sample to give sufficient acceleration of the sample to achieve good mixing. We have therefore been using a similar optical set up but initiating the reaction using caged ATP. In this device the same measurements can be made using 10-20 fold less material. This is the quantity of myosin which can be purified from a few mm of a single mammalian muscle fibre. Using these methods the following measurements can be made: rate of ATP induced dissociation of actomyosin, the affinity of ADP for actomyosin, the rate of ADP release from actomyosin and the affinity of actin for myosin and myosin ADP.

Cooperativity and regulation of scallop myosin and myosin fragments.

Scallop heavy meromyosin (HMM) preparation obtained by a new improved method showed a Mg-ATPase activity that was activated 15-fold by calcium. The ATPase activity depended on ionic strength and reached maximum at 0.1 M without altering calcium sensitivity. The highly regulated HMM and myosin preparations showed cooperative properties not seen with unregulated subfragment 1 (S1). ATPase activity of myosin and HMM increased steeply with calcium concentration, yielding Hill coefficients about 3 and 4, respectively. Calcium binding by HMM and myosin became cooperative in the presence of ADP, AMP-PNP, or ADP.Vi yielding Hill coefficients of 1.8 and 2.8, respectively. Binding of calcium by HMM in the presence of ATP was also cooperative at physiological ionic strength, whereas at low ionic strength the data fit best to a simple binding curve. In contrast, calcium binding by unregulated S1 followed a normal binding curve and was not affected by the presence of nucleotide analogues. Calcium decreased the affinity of ADP and ADP-PNP to myosin and HMM, but had no effect on the nucleotide binding to S1. The results indicate that communication between the nucleotide and calcium binding sites requires the presence of two heads and exists only in the "off" state. We propose that in the presence of calcium, interaction between the two heads is disrupted and they act independently.

Biochemical characterization and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia.

Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by N(G)-nitro-L-arginine (L-NNA). Proteins purified by 2',5'-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+- and NADPH-dependent NOS activity, which was partially inhibited by EGTA and L-NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue.

Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides.

De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase. An important feature of this inhibition is the fact that certain synergistic nucleotide pairs give more than additive inhibition. The physiological importance of synergism is in amplifying regulation by the adenine and guanine nucleotide end products of de novo synthesis. Using a new method to quantitate synergism, ADP plus GMP were confirmed [Meyer, E., and Switzer, R. L. (1978) J. Biol. Chem. 254, 5397-5402] to give strong synergistic inhibition of Bacillus subtilis glutamine PRPP amidotransferase. An X-ray structure of the ternary enzyme.ADP.GMP complex established that ADP binds to the allosteric A site and GMP to the catalytic C site. GMP increased the binding affinity of ADP for the A site by approximately 20-fold. Synergism results from a specific nucleotide-nucleotide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. Furthermore, synergism is enhanced by the competition between nucleotide inhibitor and PRPP substrate for the C site. Purine base specificity results from a backbone carbonyl interaction of Lys305' with the 6-NH2 group of adenine in the A site and a Ser347 Ogamma interaction with the 2-NH2 group of guanine in the C site. Steric considerations favor binding of the nucleoside diphosphate to the A site. Site-directed replacements of key residues increased the nucleotide concentrations needed for 50% inhibition and in some cases perturbed synergism. Mutations in either of the nucleotide sites perturbed function at both sites, supporting the important role of synergism.

The effect of ionic strength and specific anions on substrate binding and hydrolytic activities of Na,K-ATPase.

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP3-, eosine2-, p-nitrophenylphosphate, and V(max) for Na,K-ATPase and K+-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0-0.5 M of NaCl, Na2SO4, and NaNO3 and in 0.1 M Na-acetate, NaSCN, and NaClO4. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl approximately Na2SO4 approximately Na-acetate < NaNO3 < NaSCN < NaCl04. We treated the influence of NaCl and Na2SO4 on K(diss) for E x ADP as a "pure" ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO3- effect was compatible with competitive binding of NO3- and ADP in addition to the general I-effect. K(diss) for E x NO3 was approximately 32 mM. Analysis of V(max)/K(m) for Na,K-ATPase and K+-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.

GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism.

The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8-[4-[(N'-methylanthraniloyl)amino]butyl]-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes. The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation. The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.

Interacting Head Mechanism of Microtubule-Kinesin ATPase

Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10(4) M(-1)). Rate constants of dissociation of mant ADP are <1 s(-1) for the high affinity site and 75-100 s(-1) for the low affinity site for MtK379. For MtK332, the effective rate constant is 200-300 s(-1). It is proposed that the two heads of the dimer are different through the interaction with the microtubule, a strongly bound head with low affinity for 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mant ADP), similar to the single strongly bound head of the monomer and a weakly bound or detached head with high affinity for mant ADP. Rate of binding of mant ADP gave an "S"-shaped dependence on concentration for MtK379 and a hyperbolic dependence for MtK332. Binding of K379 x mant ADP dimer to microtubules releases only one mant ADP at a rate of 50 s(-1). The second strongly bound mant ADP is released by binding of nucleotides to the other head. Rates are 100 s(-1) for ATP, 30 s(-1) for AMPPNP or ATPgammaS, and 2 s(-1) for ADP. The rate of binding of mant ATP to MtK379 showed an "S"-shaped concentration dependence and limiting rate at zero concentration is <1 s(-1) while MtK332 gave a hyperbolic dependence and limiting rate of 100 s(-1). The limiting rate is determined by the rate of dissociation of mant ADP in the hydrolysis cycle. The evidence is consistent with an interacting site model in which binding of ATP to one head is required for release of ADP from the other head in the hydrolysis cycle. This model, in which the cycles are maintained partly out of phase, is an extension of the alternating site model of Hackney (Hackney, D. D. (1994) Proc. Nat. Acad. Sci. U.S.A. 91, 6865-6869). It provides a basis for a processive mechanism.

FSBA modifies both α- and β-subunits of F 1 specifically and can be bound together with AXP at the same α-subunit

Binding of 1 mole 5′-fluorosulfonylbenzoyladenosine (FSBA) per mol F 1 induces about 50% inhibition of ATPase activity and 80% inhibition of ITPase activity. The binding of additional ligand results in a further inhibition of both activities. Maximally 5 mol/mol F 1, causing complete inhibition of activity, can be bound. Using radioactive FSBA more label is found on α-subunits than on β-subunits under the usual buffer conditions. The modified amino acids are α-Tyr300, α-Tyr244 and β-Tyr368. Binding of FSBA, at least up to 3 mol/mol F 1, does not result in loss of bound ADP, whether the starting enzyme contains 2, 3 or 4 bound nucleotides. Added adenine nucleotides compete with FSBA only for binding that results in modification of β-subunits, shifting the α/ β ratio of bound label to higher values. It is concluded that the α-subunits contain two hydrophobic pockets for the binding of nucleoside moieties, with a different orientation relative to the P-loop. One pocket contains α-Tyr244 and α-Tyr300, the other β-Tyr368. Since, however, in the binding of adenine nucleotide di- or triphosphates the P-loop is involved, only one of these ligands can bind per subunit. The previously not understood binding characteristics of several substrate analogues have now become interpretable on the assumption that also the structurally homologous β-subunits contain 2 pockets where nucleoside moieties can bind. The kinetic effects of FSBA binding indicate that the first FSBA binds at the regulatory site that has a high affinity for ADP and pyrophosphate. Binding of pyrophosphate at this high-affinity regulatory site increases the V max of the enzyme, while binding at a second regulatory site, a low-affinity site, increases the rate of binding of FSBA with a factor of about 3. Binding of bicarbonate at this latter site is responsible for the disappearance of the apparent negative cooperativity of the ATPase activity.