High Affinity ATP Binding Site Research Articles

Osmotic and ionic regulation, and kinetic characteristics of a posterior gill (Na+, K+)-ATPase from the blue crab Callinectes danae on acclimation to salinity challenge

Salt tolerance reflects ecophysiological adaptation, and the wide-ranging distribution of the Brachyura mirrors their ability to adjust body fluid concentrations. The gill (Na+, K+)-ATPase underpins such hyper/hypo-regulatory mechanisms. We evaluate osmotic and chloride regulation in Callinectes danae after 10 days acclimation to a wide salinity range (5–50 ‰S), accompanying alterations in hemolymph osmolality and [Cl−] during hypo- (15 ‰S) or hyper- (45 ‰S) osmotic challenge. Further, we investigate posterior gill (Na+, K+)-ATPase kinetics, α-subunit immunolocalization and its mRNA and protein expression (15, 30 and 40 or 45 ‰S). The crab is a moderate, asymmetrical hyper/hypo-osmoregulator but is a strong, asymmetrical hyper/hypo-chloride regulator. Hyper-regulation at low salinity is sustained by a threefold increase in (Na+, K+)-ATPase activity, a 3.5-fold increase in α-subunit mRNA expression and 1.6-fold increase in protein expression. α-Subunit signal is highest in 15 ‰S-acclimated crabs, and is uniformly distributed throughout the ionocytes and pillar cells. Activity in 30- and 40% S-acclimated crabs is similar. Affinity for ATP and Na+ increases on high salinity acclimation but decreases for ouabain. K+ apparent affinity is independent of salinity, while that for Mg2+ decreases and for NH4+ increases with increasing salinity. A high-affinity ATP-binding site disappears on acclimation at any salinity. FOF1- and Na+- or K+-ATPase activities decrease with increased salinity. Hemolymph chloride hypo-regulation depends little on gill (Na+, K+)-ATPase activity. Hyper-, and hypo-osmotic and ionic regulatory capabilities in C. danae are intricate physiological processes underpinned by multifarious gill (Na+, K+)-ATPase kinetics and altered mRNA and protein expressions.

Osmotic and ionic regulation, and modulation by protein kinases, FXYD2 peptide and ATP of gill (Na+, K+)-ATPase activity, in the swamp ghost crab Ucides cordatus (Brachyura, Ocypodidae).

We analyzed the modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+-calmodulin-dependent kinase, of gill (Na+, K+)-ATPase activity in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. Osmotic and ionic regulatory ability and gill (Na+, K+)-ATPase activity also were evaluated. (Na+, K+)-ATPase activity is stimulated by exogenous pig kidney FXYD2 peptide, while phosphorylation by endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase inhibits activity. Stimulation by FXYD2 and inhibition by protein kinase C and Ca2+/calmodulin-dependent kinase are salinity-dependent. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. At low salinities, the (Na+, K+)-ATPase exhibited a single, low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18‰S, a second, cooperative, high affinity ATP-binding site appeared, corresponding to 10-20% of total (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper-/hypo-regulated in crabs acclimated at 2 to 35‰S. Cl- was well hyper-/hypo-regulated although Na+ much less so, becoming isonatremic at elevated salinity. (Na+, K+)-ATPase activity was greatest in isosmotic crabs (26‰S), decreasing notably at 35‰S and also diminishing progressively from 18to 2‰S. Hyper-osmoregulation in U. cordatus showed little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of enzymatic and osmoregulatory adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment.

Biochemical Characterization and Allosteric Modulation by Magnesium of (Na+, K+)-ATPase Activity in the Gills of the Red Mangrove Crab Goniopsis cruentata (Brachyura, Grapsidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004mmol L-1) and high (VM = 153.4 ± 7.7nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5nmolPimin-1mg-1 protein, K0.5 = 0.16 ± 0.01mmol L-1), K+ (VM = 485.3 ± 24.3nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05mmolL-1), Na+ (VM = 425.0 ± 23.4nmol Pi min-1mg-1 protein, K0.5 = 5.1 ± 0.3mmol L-1) and NH4+ (VM = 497.9 ± 24.9nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5mmolL-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8µmolL-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.

Virtual Screening and Molecular Descriptor Analysis on Dietary Phytochemicals Against Heat Shock Protein 90 Enzyme

Cancer is a disease involving unregulated cell growth and grows uncontrollably forming malignant tumors, and invades nearby parts of the body. There are over 200 different known cancers that afflict humans. The chances of surviving this disease vary by the type and location. In 2007, cancer caused about 13% of all human deaths worldwide (7.9 million). In addition, various dietary phytochemicals having anticancer activity have been reported by certain in vivo and in vitro studies. The present work focus on the molecular docking simulation studies of a set of 32 dietary phytochemicals present in fruits, vegetables, legumes, whole grains and nuts reported of having anticancer property against Heat shock protein 90 (Hsp90). Hsp90 is an important promoter of oncogene addition and cancer cell survival. It is also secreted in large quantity and found on the surface of cancer cells. The main objective of this work involves the molecular docking simulation analysis and molecular descriptors analysis of the phytochemicals where the inhibitors showed competitive inhibition with ATP at the high-affinity ATP-binding site of Hsp90 enzyme. Thus preventing the ATP binding which eventually will block the function of this enzyme. Further, the docked analogues were screened for BioAssay activity at the NCBI PubChem database revealing three of the docked phytochemicals possessing strong anti cancer property. Lastly, these three phytochemicals were evaluated for ADME-Toxicity parameters. Keywords: Hsp90, Cancer, ATP, Molecular docking, Molecular descriptors, Phytochemicals.

Structural basis of HIV-1 resistance to AZT by excision

Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

Na +, K +-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

This investigation provides an extensive characterization of the modulation by ATP, Mg 2+, Na +, K + and NH 4 + of a gill microsomal (Na +,K +)-ATPase from Callinectes danae acclimated to 15‰ salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K + modulated by NH 4 +, discussed regarding NH 4 + excretion in benthic marine crabs. The (Na +,K +)-ATPase hydrolyzed ATP at a maximum rate of 298.7 ± 16.7 nmol Pi min − 1 mg − 1 and K 0.5 = 174.2 ± 9.8 mmol L − 1 , obeying cooperative kinetics ( n H = 1.2). Stimulation by sodium ( V = 308.9 ± 15.7 nmol Pi min − 1 mg − 1 , K 0.5 = 7.8 ± 0.4 mmol L − 1 ), magnesium (299.2 ± 14.1 nmol Pi min − 1 mg − 1 , K 0.5 = 767.3 ± 36.1 mmol L − 1 ), potassium (300.6 ± 15.3 nmol Pi min − 1 mg − 1 , K 0.5 = 1.6 ± 0.08 mmol L − 1 ) and ammonium ( V = 345.1 ± 19.0 nmol Pi min − 1 mg − 1 , K 0.5 = 6.0 ± 0.3 mmol L − 1 ) ions showed site–site interactions. Ouabain inhibited (Na +,K +)-ATPase activity with K I = 45.1 ± 2.5 μmol L − 1 , although affinity for the inhibitor increased ( K I = 22.7 ± 1.1 μmol L − 1 ) in 50 mmol L − 1 NH 4 +. Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F 0F 1- and K +-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other.

Inhibition of Msh6 ATPase Activity by Mispaired DNA Induces a Msh2(ATP)-Msh6(ATP) State Capable of Hydrolysis-Independent Movement along DNA

The Msh2-Msh6 heterodimer plays a key role in the repair of mispaired bases in DNA. Critical to its role in mismatch repair is the ATPase activity that resides within each subunit. Here we show that both subunits can simultaneously bind ATP and identify the Msh6 subunit as containing the high-affinity ATP binding site and Msh2 as containing a high-affinity ADP binding site. Stable binding of ATP to Msh6 causes decreased affinity of Msh2 for ADP, and binding to mispaired DNA stabilized the binding of ATP to Msh6. Our results support a model in which mispair binding encourages a dual-occupancy state with ATP bound to Msh6 and Msh2; this state supports hydrolysis-independent sliding along DNA.

Nucleotide exchange from the high-affinity ATP-binding site in SecA is the rate-limiting step in the ATPase cycle of the soluble enzyme and occurs through a specialized conformational state.

We have characterized the kinetic and thermodynamic consequences of adenine nucleotide interaction with the low-affinity and high-affinity nucleotide-binding sites in free SecA. ATP binds to the hydrolytically active high-affinity site approximately 3-fold more slowly than ADP when SecA is in its conformational ground state, suggesting that ATP binding probably occurs when the enzyme is in another conformational state during the productive ATPase/transport cycle. The steady-state ATP hydrolysis rate is equivalent to the rate of ADP release from the high-affinity site under a number of conditions, indicating that this process is the rate-limiting step in the ATPase cycle of the free enzyme. Because efficient protein translocation requires at least a 100-fold acceleration in the ATPase rate, the rate-limiting process of ADP release from the high-affinity site is likely to play a controlling role in the conformational reaction cycle of SecA. This release process involves a large enthalpy of activation, suggesting that it involves a protein conformational change, and two observations indicate that this conformational change is different from the well-characterized endothermic conformational transition believed to gate the binding of SecA to SecYEG. First, nucleotide binding to the low-affinity site strongly inhibits the endothermic transition but does not reduce the rate of ADP release. Second, removal of Mg(2+) from an allosteric binding site on SecA does not perturb the endothermic transition but produces a 10-fold acceleration in the rate of ADP release. These divergent effects suggest that a specialized conformational transition mediates the rate-limiting ADP-release process in SecA. Finally, ADP, 2'-O-(N-methylanthraniloyl)-adenosine-5'-diphosphate (MANT-ADP), and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) bind with similar affinities to the high-affinity site and also to the low-affinity site as inferred from their consistent effects in inhibiting the endothermic transition. In contrast, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) shows 100-fold weaker affinity than ADP for the high-affinity site and no detectable interaction with the low-affinity site at concentrations up to 1 mM, suggesting that this nonhydrolyzable analogue may not be a faithful mimic of ATP in its interactions with SecA.

Replacement of Several Single Amino Acid Side Chains Exposed to the Inside of the ATP-binding Pocket Induces Different Extents of Affinity Change in the High and Low Affinity ATP-binding Sites of Rat Na/K-ATPase

To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na(+)/K(+)-ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na(+)/K(+)-ATPase alpha-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na(+)/K(+)-ATPase or low affinity ATP inhibition of K(+)-para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K(0.5). However, both the affinities for the K480E were reduced to approximately 1/20. The turnover number of the Na(+)/K(+)-ATPase and the apparent affinity for Na(+) and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K(+)-pNPPase and the apparent affinity for K(+) were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.

Hepatitis C virus NS3 ATPase/helicase: an ATP switch regulates the cooperativity among the different substrate binding sites.

The protease/helicase NS3 is believed to play a central role in the replication cycle of the hepatitis C virus (HCV), and, therefore, it is an attractive target for antiviral chemotherapy. Several enzymological studies and crystallographic structures are available for the NS3 protease and helicase domains individually, but less is known about the NTPase and helicase activities of the full-length protein. The aim of our study was to characterize from an enzymological point of view the mechanism of interaction of the full-length NS3 protease/helicase with its nucleic acid (NA) and ATP substrates. Our kinetic analysis revealed that both the NA and ATP substrates can interact cooperatively with the enzyme through the coordinated action of two binding sites. Moreover, the observation of a reciprocal influence of both substrates on the kinetics of their interaction with the enzyme suggested that the NS3 helicase works as a dimer which can exist in three functionally different states: (i) an unbound state, with two equivalent low-affinity binding sites for ATP, which shows cooperative high-affinity NA binding; (ii) an ATP-bound state, with two equivalent low-affinity NA binding sites; and (iii) a NA-bound state, with two equivalent high-affinity ATP binding sites. The cycling between these different conformational states is thus regulated by an ATP switch. These results are discussed in light of the current models for NA unwinding by the HCV NS3 helicase.

ATP Induces a Conformational Change in Lipid-bound Cytochrome c

Resonance energy transfer studies using a pyrene-labeled phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol (donor) and the heme (acceptor) of cytochrome c (cyt c) have indicated that ATP causes changes in the conformation of the lipid-bound protein (Rytömaa, M., Mustonen, P., and Kinnunen, P. K. J. (1992) J. Biol. Chem. 267, 22243-22248). Accordingly, after binding cyt c via its so called C-site to neat phosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, further quenching of donor fluorescence is caused by ATP, saturating at 2 mm nucleotide. ATP-induced conformational changes in liposome-associated cyt c could be directly demonstrated by CD in the Soret band region (380-460 nm). The latter data were further supported by time-resolved spectroscopy using the fluorescent cyt c analog with a Zn(2+)-substituted heme moiety. A high affinity ATP-binding site has been demonstrated in cyt c (Craig, D. B., and Wallace, C. J. A. (1993) Protein Sci. 2, 966-976) that is compromised by replacing the invariant Arg(91) to norleucine. Although no major effects on conformation and function of cyt c were concluded due to the modification, a significantly reduced effect by ATP on the lipid-bound [Nle(91)]cyt c was evident, implying that this modulation is mediated via the Arg(91)-containing binding site.

Binding of ATP to the Fructose-2,6-bisphosphatase Domain of Chicken Liver 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase Leads to Activation of Its 6-Phosphofructo-2-kinase

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

Ligands Presumed to Label High Affinity and Low Affinity ATP Binding Sites Do Not Interact in an (αβ)2 Diprotomer in Duck Nasal Gland Na+,K+-ATPase, nor Do the Sites Coexist in Native Enzyme

The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same alphabeta protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each alphabeta protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITC-bound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITC-labeled enzyme, TNP-ADP and ErITC inhibit p- nitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC.

NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1

Pancreatic β-cell ATP-sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regulate glucose-induced insulin secretion. To learn more about the interaction of SUR1 with nucleotides, we examined the effect of N-ethylmaleimide (NEM) modification. Photoaffinity labeling of SUR1 with 5 μM 8-azido-[α- 32P]ATP or 8-azido-[γ- 32P]ATP was inhibited by NEM with K i of 1.8 μM and 2.4 μM, and Hill coefficients of 0.94 and 1.1, respectively. However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 μM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1.

The Anion-stimulated ATPase ArsA Shows Unisite and Multisite Catalytic Activity

ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.

Microenvironment of the High Affinity ATP-Binding Site of Na+/K+-ATPase Is Slightly Acidic

Fluorescein-5′-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per α-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P.This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (αβ)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 ± 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.

Tight Binding of Bulky Fluorescent Derivatives of Adenosine to the Low Affinity E2ATP Site Leads to Inhibition of Na+/K+-ATPase: ANALYSIS OF STRUCTURAL REQUIREMENTS OF FLUORESCENT ATP DERIVATIVES WITH A KOSHLAND-NÉMETHY-FILMER MODEL OF TWO INTERACTING ATP SITES

A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC 3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2',3'-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E1ATP site (Kd = 0.7 microM) and to the E2ATP site (Kd = 210 microM), but 3'(2')-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E1ATP binding site (Kd = 200 microM) than to the E2ATP binding site (Kd = 80 microM). 3'(2')-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (Kd = 10 microM). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na+/K+-ATPase. A number of derivatives of 2'(3')-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E2ATP site and inhibit Na+/K+-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E2ATP site.

Kinetics of Na +-Dependent Conformational Changes of Rabbit Kidney Na +,K +-ATPase

The kinetics of Na +-dependent partial reactions of the Na +,K +-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-( p-(dipentylamino)phenyl)butadienyl)pyridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/ τ 1 ≈ 155 s −1 for 5-IAF-labeled enzyme and 1/ τ 1 ≈ 200 s −1 for native enzyme (saturating [ATP] and [Na +], pH 7.4 and 24°C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E 1ATP(Na +) 3 → E 2P(Na +) 3 + ADP). The smaller amplitude slow phase, 1/ τ 2 = 30–45 s −1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K + ions (E 2P ⇔ E 2). The Na + concentration dependence of 1/ τ 1 showed half-saturation at a Na + concentration of 6–8 mM, with positive cooperativity involved in the occupation of the Na + binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/ τ 1 was 8.0 (± 0.7) μM. It was found that P 3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/ τ 1 observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.

Cloning and expression of the secA gene of a marine bacterium, Vibrio alginolyticus, and analysis of its function in Escherichia coli.

We report the cloning, sequencing and functional characterization of the secA gene of a marine bacterium, Vibrio alginolyticus, which has been suggested to utilize ATP and the sodium motive force for protein translocation. Oligodeoxynucleotides corresponding to highly conserved regions of Escherichia coli secA located in the high affinity ATP binding site were utilized as PCR primers to clone the secA gene of V. alginolyticus. It was shown to encode a 103.3-kDa protein. The deduced amino acid sequence of V. alginolyticus SecA (VaSecA) exhibits a high degree of identity (72.7%) to SecA of E. coli (EcSecA). The secA gene of E. coli forms an operon with upstream orfX, whereas no counterpart is present upstream of V. alginolyticus secA. Azide derepresses the EcSecA translation, whereas the level of VaSecA was unaffected by azide. Expression of VaSecA in E. coli carrying a temperature-sensitive secA mutation restored both growth and protein translocation at a non-permissive temperature. VaSecA was thus able to substitute for EcSecA despite the fact that the energy requirement for protein translocation differs between the two organisms. VaSecA was overproduced in V. alginolyticus and purified to homogeneity for N-terminal sequencing. The endogenous ATPase activity of the purified VaSecA was comparable with that of EcSecA. 1998 Elsevier Science B.V.

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.