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1.
The present study contributes to the problem of the dynamic structure of mitochondrial F1-ATPase and the functional interrelation of so-called tight nucleotide binding sites. Nucleotide analogs are used as a tool to differentiate two distinct functional states of the membrane-bound enzyme, proposed to reflect corresponding conformational states; they reveal F1-ATPase as a dual-state enzyme: ATP-synthetase, and ATP-hydrolase. The analogs used are 3-naphthoyl esters of AD(T)P, and 2(3)-O-trinitrophenyl ethers of AD(T)P. Both types of analogs act inversely to each other with respect to their relative effects on oxidative phosphorylation and on ATPase in submitochondrial vesicles. The respective ratios ofK
i versus both processes are 250/1 compared to 1/170. It is also shown that in the presence of the inhibitory 3-esters oxidative phosphorylation deviates from linear kinetics and that these inhibitors induce a lag time of oxidative phosphorylation depending on the initial pattern of nucleotides available to energized submitochondrial vesicles. The duration of the lag time coincides with the time course of displacement of the analog from a tight binding site. The conclusions of the study are: (a) the catalytic sites of F1-ATP-synthetase are not operating independently from each other; they rather interact in a cooperative manner; (b) F1-ATPase as a dual-state enzyme exhibits highly selective responses to tight binding of nucleotides or analogs in its energized (membrane-bound) state versus its nonenergized state, respectively.Abbreviations used: N-AD(T)P, 3-O-naphthoyl(1)-AD(T)P; DMAN-AD(T)P, 3-O-(5-dimethylaminonaphthoyl(1))-AD(T)P, also termed F-AD(T)P in previous papers because of its fluorescence; TNP-AD(T)P, 2(3)-O-(2,4,6-trinitrophenyl)-AD(T)P; FCCP,p-trifluoromethoxycarbonylcyanide phenylhydrazone. 相似文献
2.
The ATP-dependent transport enzyme known as P-glycoprotein (P-gp) confers multidrug resistance (MDR) against many unrelated drugs and xenobiotics. To understand better the broad substrate specificity of the enzyme as well as the mechanism of substrate transport out of the cell, it is critical to characterize the substrate binding sites. Since approximately 1 ATP is hydrolyzed per transport event, phosphate release rate provides a steady-state kinetics assay. Notably, the substrate H33342 causes a decrease in the baseline hydrolysis of ATP (probably due to competition for transport with an endogenous membrane lipid substrate) providing an excellent tool for a comprehensive graphical kinetic analysis of the interaction of substrate pairs at the transport site(s) allowing the determination of inhibition type and hence characterization of transport binding sites. The substrate H33342 interacted with quinidine, progesterone, and propranolol in a non-competitive manner, indicating that binding of H33342 precludes active transport of these other substrates at a distinct site. Compounds such as TPP+ and verapamil, and perhaps also nicardipine, interacted with H33342 as mixed-type inhibitors. This type of interaction results from a reduced affinity at the opposing active site by a factor of alpha and sometimes a partial activity of a fraction beta. Indeed, H33342 binding caused a roughly four-fold reduced affinity for TPP+. Using this definitive approach to inhibition kinetics, we were able to establish traits of a second transport site in P-gp. Therefore, the sites are unequal; however, the performance at one site is contingent on the other being unoccupied, and transport is also sometimes mitigated when the other site is occupied. 相似文献
3.
Moolenaar GF Herron MF Monaco V van der Marel GA van Boom JH Visse R Goosen N 《The Journal of biological chemistry》2000,275(11):8044-8050
We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads. With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP. For the binding of UvrC to the UvrB-DNA complex no cofactor is needed. The subsequent induction of 3' incision does require ATP binding by UvrB but not hydrolysis. This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5' side of the damage. In contrast, the 5' incision is not dependent on ATP binding because it occurs with the same efficiency with ADP. We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule. A DNA substrate containing damage close to the 5' end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G. F., Monaco, V., van der Marel, G. A., van Boom, J. H., Visse, R., and Goosen, N. (2000) J. Biol. Chem. 275, 8038-8043). To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound. Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed. 相似文献
4.
Guanosine triphosphate and formycin triphosphate (FTP) in the presence of excess Mg2+ can bind to empty non-catalytic sites of spinach chloroplast ATPase (CF1). This results in a greatly reduced capacity for ATP hydrolysis compared to the enzyme with non-catalytic sites filled with ATP. With two GTP bound at non-catalytic sites the inhibition is about 90%; with two FTP bound about 80% inhibition is obtained. Binding and release of the nucleotides from the non-catalytic sites are relatively slow processes. Exposure of CF1 with one or two empty non-catalytic sites to 5–10 μM FTP or GTP for 15 min suffices for about 50% of the maximum inhibition. Reactivation of CF1 after exposure to higher FTP or GTP concentrations requires long exposure to 2 μM EDTA. The findings show that, contrary to previous assumptions, GTP can bind tightly to non-catalytic sites of CF1. They suggest that the presence of adenine nucleotides at non-catalytic sites might be essential for high catalytic capacity of the F1 ATPases. 相似文献
5.
C A Hrycyna M Ramachandra U A Germann P W Cheng I Pastan M M Gottesman 《Biochemistry》1999,38(42):13887-13899
Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site. 相似文献
6.
A M Shimabuku T Nishimoto K Ueda T Komano 《The Journal of biological chemistry》1992,267(7):4308-4311
Two ATP-binding domains are found in members of the family of ATP-dependent transport proteins, which includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, full-length and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the Escherichia coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprotein beta-galactosidase showed ATPase activity with apparent specific activity of 180 nmol/mg/min, a value higher than previously reported, in the presence of phospholipids, suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain. 相似文献
7.
Mutagenesis was used to investigate the functional role of six pairs of aspartate and glutamate residues (D450/D1093, E482/E1125, E552/E1197, D558/D1203, D592/D1237, and E604/E1249) that are highly conserved in the nucleotide binding sites of P-glycoprotein (Mdr3) and of other ABC transporters. Removal of the charge in E552Q/E1197Q and D558N/D1203N produced proteins with severely impaired biological activity when the proteins were analyzed in yeast cells for cellular resistance to FK506 and restoration of mating in a ste6Delta mutant. Mutations at other acidic residues had no apparent effect in the same assays. These four mutants were expressed in Pichia pastoris, purified to homogeneity, and biochemically characterized with respect to ATPase activity. Studies with purified proteins showed that mutants D558N and D1203N retained 14 and 30% of the drug-stimulated ATPase activity of wild-type (WT) Mdr3, respectively, and vanadate trapping of 8-azido[alpha-(32)P]nucleotide confirmed slower basal and drug-stimulated 8-azido-ATP hydrolysis compared to that for WT Mdr3. The E552Q and E1197Q mutants showed no drug-stimulated ATPase activity. Surprisingly, drugs did stimulate vanadate trapping of 8-azido[alpha-(32)P]nucleotide in E552Q and E1197Q at a level similar to that of WT Mdr3. This suggests that formation of the catalytic transition state can occur in these mutants, and that the bond between the beta- and gamma-phosphates is hydrolyzed. In addition, photolabeling by 8-azido[alpha-(32)P]nucleotide in the presence or absence of drug was also detected in the absence of vanadate in these mutants. These results suggest that steps after the transition state, possibly involved in release of MgADP, are severely impaired in these mutant enzymes. 相似文献
8.
Al-Shawi MK Polar MK Omote H Figler RA 《The Journal of biological chemistry》2003,278(52):52629-52640
ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented. 相似文献
9.
Kim IW Peng XH Sauna ZE FitzGerald PC Xia D Müller M Nandigama K Ambudkar SV 《Biochemistry》2006,45(24):7605-7616
Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue approximately 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18,514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in approximately 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain approximately 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP). 相似文献
10.
ABC transporters constitute one of the most abundant membrane transporter families. The most common feature shared in the family is the highly conserved nucleotide binding domains (NBDs) that drive the transport process through binding and hydrolysis of ATP. Molecular dynamics simulations are used to investigate the effect of ATP hydrolysis in the NBDs. Starting with the ATP-bound, closed dimer of MalK, four simulation systems with all possible combinations of ATP or ADP-Pi bound to the two nucleotide binding sites are constructed and simulated with equilibrium molecular dynamics for ∼70 ns each. The results suggest that the closed form of the NBD dimer can only be maintained with two bound ATP molecules; in other words, hydrolysis of one ATP can lead to the opening of the dimer interface of the NBD dimer. Furthermore, we observed that the opening is an immediate effect of hydrolysis of ATP into ADP and Pi rather than the dissociation of hydrolysis products. In addition, the opening is mechanistically triggered by the dissociation of the LSGGQ motif from the bound nucleotide. A metastable ADP-Pi bound conformational state is consistently observed before the dimer opening in all the simulation systems. 相似文献
11.
L J Blackwell K P Bjornson D J Allen P Modrich 《The Journal of biological chemistry》2001,276(36):34339-34347
The role of MutS ATPase in mismatch repair is controversial. To clarify further the function of this activity, we have examined adenine nucleotide effects on interactions of Escherichia coli MutS with homoduplex and heteroduplex DNAs. In contrast to previous results with human MutS alpha, we find that a physical block at one end of a linear heteroduplex is sufficient to support stable MutS complex formation in the presence of ATP.Mg(2+). Surface plasmon resonance analysis at low ionic strength indicates that the lifetime of MutS complexes with heteroduplex DNA depends on the nature of the nucleotide present when MutS binds. Whereas complexes prepared in the absence of nucleotide or in the presence of ADP undergo rapid dissociation upon challenge with ATP x Mg(2+), complexes produced in the presence of ATP x Mg(2+), adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) x Mg(2+), or ATP (no Mg(2+)) are resistant to dissociation upon ATP challenge. AMPPNP x Mg(2+) and ATP (no Mg(2+)) reduce MutS affinity for heteroduplex but have little effect on homoduplex affinity, resulting in abolition of specificity for mispaired DNA at physiological salt concentrations. Conversely, the highest mismatch specificity is observed in the absence of nucleotide or in the presence of ADP. ADP has only a limited effect on heteroduplex affinity but reduces MutS affinity for homoduplex DNA. 相似文献
12.
Preincubation of submitochondrial particles with ADP in the presence of Mg2+ results in the complete inhibition of ATPase which is slowly reactivated in the assay mixture containing ATP and the ATP regenerating system. Significantly, the rate of activation increases as the concentration of ADP in the preincubation mixture rises from 1 microM to 20 microM and reaches a constant value at higher ADP concentrations. The first-order rate constant for the activation process in the assay mixture is ATP-dependent at any level of inhibitory ADP. The data obtained strongly suggest that two ADP-specific inhibitory sites and one ATP-specific hydrolytic site are present in F1-F0 ATPase. Taking into account the (3 alpha.3 beta).gamma.delta.epsilon structure of F1, it is concluded that the synchronous discharge of ADP from two inhibitory sites during the activation occurs after ATP binds to the ATPase catalytic site. 相似文献
13.
Malyan AN 《Photosynthesis research》2006,88(1):9-18
A modified ‘cold chase’ technique was used to study tight [14C]ADP and [14C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF0F1). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF0F1 or CF1 reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k
d of about 5 min−1. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF0F1. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF1 dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF1 becomes bound to a thylakoid membrane. 相似文献
14.
P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp.ADP.Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomes vis à vis the substrate, they show comparable t(12) for both incorporation and release of nucleotide, and the affinities for [alpha-(32)P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [alpha-(32)P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp. 相似文献
15.
Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding. 相似文献
16.
The role of P-glycoprotein in mediating the drug-resistance phenotype in multidrug resistant cells is now well documented. It is thought to function as an energy-dependent drug-efflux pump of broad specificity. Structurally, P-glycoprotein is an internally duplicated molecule containing two large multi-spanning transmembrane domains and two cytoplasmic ATP binding domains. In this report we demonstrate that monoclonal antibodies C219, C494, and C32 directed against short linear regions of the P-glycoprotein molecule inhibit ATP binding to P-glycoprotein in vitro. We also provide direct evidence that both predicted ATP-binding domains bind ATP and that there is co-operativity between the two sites. In addition, the capacity of P-glycoprotein to bind the calcium channel blocker, azidopine, is inhibited differentially by the antibodies. These observations are the first evidence linking specific perturbations of the P-glycoprotein molecule with ATP and drug binding. 相似文献
17.
A. N. Malyan 《Biochemistry. Biokhimii?a》2013,78(13):1512-1523
ATP synthases (FoF1-ATPases) of chloroplasts, mitochondria, and bacteria catalyze ATP synthesis or hydrolysis coupled with the transmembrane transfer of protons or sodium ions. Their activity is regulated through their reversible inactivation resulting from a decreased transmembrane potential difference. The inactivation is believed to conserve ATP previously synthesized under conditions of sufficient energy supply against unproductive hydrolysis. This review is focused on the mechanism of nucleotide-dependent regulation of the ATP synthase activity where the so-called noncatalytic nucleotide binding sites are involved. Properties of these sites varying upon free enzyme transition to its membrane-bound form, their dependence on membrane energization, and putative mechanisms of noncatalytic site-mediated regulation of the ATP synthase activity are discussed. 相似文献
18.
《Life sciences》1993,53(24):PL393-PL398
Recent pharmacological studies demonstrate that Syrian hamster melanoma (RPMI 1846) cells possess a nanomolar-affinity binding site for melatonin. Inhibition of 2-[125|]-iodomelatonin binding to RPMI membranes by melatonergic ligands exhibit a rank order relationship similar to that observed in hamster hypothalamus. Biochemical studies indicate that the melatonin binding site in RPMI 1846 cells is not coupled in either a stimulatory or inhibitory fashion to adenylate cyclase as a second messenger. We now report that stimulation of RPMI 1846 melanoma cells with melatonergic agonists induces phosphoinositide hydrolysis in a concentration-dependent manner EC50 : N-acetylserotonin = 0.29 μM; 2-l-Melatonin = 0.39 μM; 6-Cl-Melatonin = 0.38 μM; Melatonin = 0.45 μM. Further, phosphoinositide hydrolysis induced by 2-l-melatonin and N-acetylserotonin was blocked by pre-incubation with the melatonin antagonist N-acetyltryptamine and prazosin, an antagonist which exhibits potency at the nanomolar affinity melatonin binding site. 2-l-melatonin and N-acetylserotonin-induced phosphoinositide hydrolysis were not blocked by the serotonin type 2 antagonist ketanserin or alpha-adrenergic antagonist, phentolamine. These data suggest that melatonin binding sites on RPMI 1846 cells are linked to phosphoinositide hydrolysis as a second messenger. 相似文献
19.
Conventional myosin is representative of biomolecular motors in which the hydrolysis of adenosine triphosphate (ATP) is coupled to large-scale structural transitions both in and remote from the active site. The mechanism that underlies such “mechanochemical coupling,” especially the causal relationship between hydrolysis and allosteric structural changes, has remained elusive despite extensive experimental and computational analyses. In this study, using combined quantum mechanical and molecular mechanical simulations and different conformations of the myosin motor domain, we provide evidence to support that regulation of ATP hydrolysis activity is not limited to residues in the immediate environment of the phosphate. Specifically, we illustrate that efficient hydrolysis of ATP depends not only on the proper orientation of the lytic water but also on the structural stability of several nearby residues, especially the Arg238-Glu459 salt bridge (the numbering of residues follows myosin II in Dictyostelium discoideum) and the water molecule that spans this salt bridge and the lytic water. More importantly, by comparing the hydrolysis activities in two motor conformations with very similar active-site (i.e., Switches I and II) configurations, which distinguished this work from our previous study, the results clearly indicate that the ability of these residues to perform crucial electrostatic stabilization relies on the configuration of residues in the nearby N-terminus of the relay helix and the “wedge loop.” Without the structural support from those motifs, residues in a closed active site in the post-rigor motor domain undergo subtle structural variations that lead to consistently higher calculated ATP hydrolysis barriers than in the pre-powerstroke state. In other words, starting from the post-rigor state, turning on the ATPase activity requires not only displacement of Switch II to close the active site but also structural transitions in the N-terminus of the relay helix and the “wedge loop,” which have been proposed previously to be ultimately coupled to the rotation of the converter subdomain 40 Å away. 相似文献
20.
Information content of binding sites on nucleotide sequences 总被引:73,自引:0,他引:73
Repressors, polymerases, ribosomes and other macromolecules bind to specific nucleic acid sequences. They can find a binding site only if the sequence has a recognizable pattern. We define a measure of the information (R sequence) in the sequence patterns at binding sites. It allows one to investigate how information is distributed across the sites and to compare one site to another. One can also calculate the amount of information (R frequency) that would be required to locate the sites, given that they occur with some frequency in the genome. Several Escherichia coli binding sites were analyzed using these two independent empirical measurements. The two amounts of information are similar for most of the sites we analyzed. In contrast, bacteriophage T7 RNA polymerase binding sites contain about twice as much information as is necessary for recognition by the T7 polymerase, suggesting that a second protein may bind at T7 promoters. The extra information can be accounted for by a strong symmetry element found at the T7 promoters. This element may be an operator. If this model is correct, these promoters and operators do not share much information. The comparisons between R sequence and R frequency suggest that the information at binding sites is just sufficient for the sites to be distinguished from the rest of the genome. 相似文献