A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP.

We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P(i)) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site.     

The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump.

The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.     

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.     

Real-time measurement of myosin-nucleotide noncovalent complexes by electrospray ionization mass spectrometry

Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 +/- 10 Da were obtained with the binding of ADP to the active site and by 530 +/- 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of approximately 1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 muM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of MgATP in the activation and reassociation of cAMP-dependent protein kinase I: consequences of replacing the essential arginine in cAMP binding site A.

The type I form of cAMP-dependent protein kinase binds MgATP with a high affinity, and binding of MgATP decreases the affinity of the holoenzyme for cAMP [Hofmann et al. (1975) J. Biol. Chem. 250, 7795]. Holoenzyme was formed here with a mutant form of the bovine recombinant type I regulatory subunit where the essential arginine in site A, Arg-209, was replaced with Lys. Although this mutation does not significantly change the high-affinity binding of MgATP to the holoenzyme, it does abolish high-affinity binding of cAMP to site A. In the absence of MgATP, binding of cAMP to site B is sufficient to promote dissociation of the holoenzyme complex and activation of the catalytic subunit [Bubis et al. (1988) J. Biol. Chem. 263, 9668]. In the presence of MgATP however, holoenzyme formed with this mutant regulatory subunit is very resistant to cAMP. The Kd(cAMP) was greater than 1 microM, and the Ka(cAMP) increased 60-fold from 130 nM to 6.5 microM in the presence of MgATP. Thus, MgATP serves as a lock that selectively stabilizes the holoenzyme and inhibits activation. Both site A and site B are shielded from cAMP in the presence of MgATP. These results suggest that Arg-209 may play a role in stabilizing the MgATP.holoenzyme complex in addition to its role in binding the exocyclic oxygens of cAMP when cAMP is bound to the regulatory subunit. The catalytic subunit also reassociates rapidly with this mutant regulatory subunit, and in contrast to the wild-type regulatory subunit, holoenzyme formation does not require MgATP.     

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.     

Intramolecular cross-linking at the active site of the Ca2+-ATPase of sarcoplasmic reticulum. High and low affinity nucleotide binding and evidence of active site closure in E2-P

Limited reaction of glutaraldehyde with the Ca2+-ATPase (Mr approximately 110,000) of sarcoplasmic reticulum results in intramolecular cross-linking at the active site, which can be detected by an anomalous increase in apparent molecular weight (Mr approximately 125,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ross D.C., and McIntosh D.B. (1987) J. Biol. Chem. 262, 2042-2049). ATP, ADP, AMPPCP, trinitrophenyladenosine triphosphate, and decavanadate inhibited the cross-link in a manner suggestive of a homogeneous class of inhibitory sites, with K0.5 values for inhibition in agreement with Kd values for binding to the active site. Cross-link formation was inhibited in proportion to phosphoenzyme levels formed from Pi (E2-P) whereas stoichiometric phosphorylation from CaATP (E1-P) had no effect. Inhibition was observed at millimolar concentrations of CaATP, indicative of nucleotide binding to E1-P. MgATP, in the presence of Ca2+, inhibited cross-linkage in the micromolar and millimolar concentration ranges, the former attributable to E1 X ATP and E2-P formation and the latter to ATP binding mainly to E1-P. The inability to cross-link the active site only of the E2-P intermediate suggests a unique active site conformation, possibly a closed active site cleft, which we suggest is linked to low affinity, inwardly orientated Ca2+-binding sites.     

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.     

The Escherichia coli PriA helicase has two nucleotide-binding sites differing dramatically in their affinities for nucleotide cofactors. 1. Intrinsic affinities, cooperativities, and base specificity of nucleotide cofactor binding

Interactions of the Escherichia coli PriA helicase with nucleotide cofactors have been studied using the fluorescence titration and analytical ultracentrifugation techniques. Binding of unmodified cofactors was characterized by the fluorescence competition titration method. The obtained data establish that at saturation the PriA helicase binds two nucleotide molecules per protein monomer. This result corroborates with the primary structure of the protein, which contains sequence motifs implicated as putative nucleotide-binding sites. The intrinsic affinities of the binding sites differ by 2-4 orders of magnitude. Thus, the PriA helicase has a strong and a weak nucleotide-binding site. The binding sites differ dramatically in their properties. The strong site is highly specific for adenosine cofactors, while the weak site shows very modest base specificity. The affinities of the strong and weak binding sites for ATP are lower than the affinities for ADP, although both sites have similar affinity for the inorganic phosphate group. Unlike the weak site, the affinity of the strong site profoundly depends on the structure of the phosphate group of the ATP cofactor. Binding of unmodified nucleotides indicates the presence of positive cooperative interactions between bound cofactors (i.e., the existence of communication between the two sites). Magnesium cations are specifically involved in controlling the cofactor affinity for the strong site, while the affinity of the weak site is predominantly determined by interactions between the phosphate group and ribose regions of the cofactor and the protein matrix. The significance of these results for the activities of the PriA helicase is discussed.     

Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

The amino terminus regulates binding to and activation of cGMP-dependent protein kinase

The allosteric regulation of binding to and the activation of cGMP-dependent protein kinase (cGMP kinase) was studied under identical conditions at 30 degrees C using three forms of cGMP-kinase which differed in the amino-terminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4 +/- 0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino-terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP-binding sites, Km values for MgATP and the heptapeptide kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrations of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 microM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino-terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP-dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino-terminal domain.     

Expression and characterization of the N- and C-terminal ATP-binding domains of MRP1

The His(6)-tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized. Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective. Both MRP1 nucleotide binding domains showed ATPase activities (V(max) values between 5-10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter. The K(M) value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K(M) value of the native or the purified and reconstituted transporter, N-ethylmaleinimide and A1F(4) inhibited the ATPase activity of both nucleotide binding domains.     

Further characterization of nucleotide binding sites on chloroplast coupling factor one

The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.     

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver. Factors affecting the activation of the binding protein by adenosine 5'-triphosphate.

A cyclic AMP-adenosine binding protein, whose binding sites are activated by preincubation in the presence of Mg⁺-ATP, has been purified to apparent homogeneity from mouse liver (P.M. Ueland and S.O. Døskeland, 1977, J. Biol. Chem.,252, 677–686). The degree of activation of both the cyclic AMP binding site and a high-affinity site for adenosine depends on the concentration of ATP during the preincubation. The velocity and the degree of activation are dependent on the temperature and the presence of Mg²⁺ and K⁺. The NH₄⁺ ion can be substituted for K⁺, whereas Na⁺ is inefficient. Low pH promotes the conversion from the inactive to the active form. The apparent affinity for adenosine to the high-affinity site for this adenine derivative and the affinity for cyclic AMP to the site specific for this nucleotide are independent of the degree of activation as judged from the slope of Scatchard plots. The activation of the cyclic AMP binding site by ATP (6 mm) was determined at pH 7 in the presence of 10 μm cyclic AMP, AMP, ADP, or adenosine. Adenosine specifically inhibits the activation and does not promote the inactivation of the binding protein. The possibility that the apparent inhibition of activation was effected by interference with cyclic AMP binding by adenosine was ruled out.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.     

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.