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Substrate Binding (substrate + binding)
Terms modified by Substrate Binding Selected AbstractsPromiscuous Substrate Binding Explains the Enzymatic Stereo- and Regiocontrolled Synthesis of Enantiopure Hydroxy Ketones and DiolsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-12 2009Marcela Kurina-Sanz Abstract Regio- and stereoselective reductions of several diketones to afford enantiopure hydroxy ketones or diols were accomplished using isolated alcohol dehydrogenases (ADHs). Results could be rationalised taking into account different (promiscuous) substrate-binding modes in the active site of the enzyme. Furthermore, interesting natural cyclic diketones were also reduced with high regio- and stereoselectivity. Some of the 1,2- and 1,3-diketones used in this study were reduced by employing a low excess of the hydrogen donor (2-propanol) due to the quasi-irreversibility of these ADH-catalysed processes. Thus, using lower quantities of co-substrate, scale-up could be easily achieved. [source] The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitiveFEBS JOURNAL, Issue 20 2004Linda O'Reilly Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the ,-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures. [source] Structure,Activity Relationship Studies in Single-Site Esterase Peptide DendrimersISRAEL JOURNAL OF CHEMISTRY, Issue 1 2009Sacha Javor We recently reported on peptide dendrimers with a single catalytic site at the dendrimer core catalyzing the hydrolysis of acetoxy- and butyryloxy-pyrene trisulfonate 1a/b in aqueous buffer with Michaelis,Menten kinetics. Substrate binding is mediated by a pair of protonated arginine or histidine residues in the first generation branch, and esterolysis is performed by the imidazole side-chain of a histidine residue in the core acting as a general base or nucleophile. Herein we report on a structure,activity relationship study searching for an optimal combination between amino acid sequence and catalytic machinery. Installation of histidine residues onto the aromatic dendrimer framework "R" leads to 10-fold higher rate acceleration up to kcat/kuncat = 1.5 * 103 at pH 5.5 with dendrimers RG3H (AcYT)8 (BWG)4 (BHS)2BHS and RMG3H (AcYT)8(BWG)4(BHSG)2BHS (one-letter codes for L -amino acids; Ac = acetyl, B = L -2,3-diaminopropionic acid branching point, C-terminus is amide -CONH2). These dendrimers reach the compactness of a native folded protein. [source] Substrate binding induces structural changes in cytochrome P450camACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Keisuke Sakurai The binding of (+)-camphor to cytochrome P450cam (P450cam) expels a cluster of waters at the active site, raising the redox potential of the haem to an extent that allows reduction by the electron-transfer system. This binding was reported to involve no significant structural changes in the protein. Here, two ferric P450cam structures partially complexed with (+)-camphor were determined by X-ray crystallography at 1.30,1.35,Å resolution, revealing the structures of the substrate-free and substrate-bound forms. (+)-Camphor binding induces rotation of Thr101 to form a hydrogen bond that acts as a hydrogen donor to a peripheral haem propionate. This bonding contributes to the redox-potential change. [source] Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor treeFEBS JOURNAL, Issue 22 2001Liang Xie Cinnamomin is a new type II ribosome-inactivating protein (RIP). Its A-chain exhibits RNA N -glycosidase activity to inactivate the ribosome and thus inhibit protein synthesis, whereas the glycosylated B-chain is a lectin. The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the nucleotide sequences of cDNAs of cinnamomin A- and B-chains. It is composed of a total of 549 amino-acid residues: 271 residues in the A-chain, a 14-residue linker and 264 residues in the B-chain. To explore its biological function, the cinnamomin A-chain was expressed in Escherichia coli with a yield of 100 mg per L of culture, and purified through two-step column chromatography. After renaturation, the recovery of the enzyme activity of the expressed A-chain was 80% of that of native A-chain. Based on the modeling of the three-dimensional structure of the A-chain, the functional roles of five amino acids and the only cysteine residues were investigated by site-directed mutagenesis or chemical modification. The conserved single mutation of the five amino-acid residues led to 8,50-fold losses of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N -glycosidase activity of the A-chain. Most interestingly, the strong electric charge introduced at the position of the single cysteine in A-chain seemed to play a role in enzyme/substrate binding. [source] Insights into the anthrax lethal factor,substrate interaction and selectivity using docking and molecular dynamics simulationsPROTEIN SCIENCE, Issue 8 2009Georgios A. Dalkas Abstract The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn-dependent, highly specific metalloprotease with a molecular mass of ,90 kDa that cleaves most isoforms of the family of mitogen-activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK-based synthetic peptide provided structure-activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF-MEK/MKK interaction along the catalytic channel up to a distance of 20 Å from the zinc atom. This residue-specific view of the enzyme-substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot-spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction. [source] Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltoseFEBS JOURNAL, Issue 20 2010Keizo Yamamoto The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (,/,)8 -barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310,315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively. [source] Bacitracin is not a specific inhibitor of protein disulfide isomeraseFEBS JOURNAL, Issue 11 2010Anna-Riikka Karala To successfully dissect molecular pathways in vivo, there is often a need to use specific inhibitors. Bacitracin is very widely used as an inhibitor of protein disulfide isomerase (PDI) in vivo. However, the specificity of action of an inhibitor for a protein-folding catalyst cannot be determined in vivo. Furthermore, in vitro evidence for the specificity of bacitracin for PDI is scarce, and the mechanism of inhibition is unknown. Here, we present in vitro data showing that 1 mm bacitracin has no significant effect on the ability of PDI to introduce or isomerize disulfide bonds in a folding protein or on its ability to act as a chaperone. Where bacitracin has an effect on PDI activity, the effect is relatively minor and appears to be via competition of substrate binding. Whereas 1 mm bacitracin has minimal effects on PDI, it has significant effects on both noncatalyzed protein folding and on other molecular chaperones. These results suggest that the use of bacitracin as a specific inhibitor of PDI in cellular systems requires urgent re-evaluation. [source] Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HIIFEBS JOURNAL, Issue 19 2008Muhammad S. Rohman Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi,Goutières syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P, were constructed. All mutant proteins were less stable than the wild-type protein by 2.9,7.6 °C in Tm. A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable. [source] Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis, implication of necessity for enzyme propertiesFEBS JOURNAL, Issue 9 2008Hsu-Han Chuang The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, kcat/Km, was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl- N-N,-diacetyl chitobiose or 4-methylumbelliferyl- N - N,- N,-triacetyl chitotriose or insoluble ,-chitin substrate. By contrast, changes to substrate affinity, Km, and turnover rate, kcat, varied considerably for both types of chitin substrates between the full-length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full-length enzyme. The truncated mutants retained similar substrate-binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full-length chitinase molecule. Fluorescence spectroscopy indicated that both C-terminal deletion mutants retained an active folding conformation similar to the full-length enzyme. However, a CD melting unfolding study was able to distinguish between the full-length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C-terminal chitin-binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C-terminal chitin-binding region and the fibronectin (III) motif. [source] Crystal structure of archaeal highly thermostable L -aspartate dehydrogenase/NAD/citrate ternary complexFEBS JOURNAL, Issue 16 2007Kazunari Yoneda The crystal structure of the highly thermostable l -aspartate dehydrogenase (l -aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidusl -aspDH was refined at a resolution of 1.9 Å with a crystallographic R -factor of 21.7% (Rfree = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidusl -aspDH/NAD/citrate ternary complex and the Thermotoga maritimal -aspDH/NAD binary complex showed that A. fulgidusl -aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritimal -aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidusl -aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two l -aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to l -aspDH and of the molecular basis of the high thermostability of a hyperthermophilic l -aspDH. [source] Three mammalian cytochromes b561 are ascorbate-dependent ferrireductasesFEBS JOURNAL, Issue 16 2006Dan Su Cytochromes b561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b561 (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b) were expressed in a Saccharomyces cerevisiae,fre1,fre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce ferric iron (Fe3+). The expression of each of these cytochromes b561 was able to rescue the growth defect of the ,fre1,fre2 mutant cells in iron-deficient conditions, suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b561 showed significant FeCN and Fe3+ -EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site-directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated, mutations of four His residues (H47, H83, H117 and H156), one Tyr (Y66) and one Arg (R67) completely abrogated the FeCN reductase activity, whereas mutations of Arg (R149), Phe (F44), Ser (S115), Trp (W119), Glu (E196), and Gln (Q131) affected the ferrireductase activity to some degree. These mutations may affect the heme coordination, ascorbate binding, and/or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This study demonstrates the ascorbate-dependent transmembrane ferrireductase activities of members of the mammalian cytochrome b561 family of proteins. [source] Recombinant human glucose-6-phosphate dehydrogenaseFEBS JOURNAL, Issue 14 2002Evidence for a rapid-equilibrium random-order mechanism Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver,Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 µm, close to the value of 6.8 µm calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding. [source] Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycleFEBS JOURNAL, Issue 17 2001Shin-ichi Yokota The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of ,, , and ,-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the , and , subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower , and , subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the , and , subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of , and , subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells. [source] Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetryFEBS JOURNAL, Issue 15 2001Yi Liang Thermodynamics of the refolding of denatured d -glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone,substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy,entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, ,156 kJ·mol,1·K,1, at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0,25.0 °C. With increasing temperature from 15.0 to 37.0 °C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 °C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected. [source] Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutaseFEBS JOURNAL, Issue 24 2000Elucidation of the roles of amino acids involved in substrate binding, catalysis The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm::HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques. [source] The hemopexin domain of MMP-9 inhibits angiogenesis and retards the growth of intracranial glioblastoma xenograft in nude miceINTERNATIONAL JOURNAL OF CANCER, Issue 2 2009Ravesanker Ezhilarasan Abstract Matrix Metalloproteinase-9 (MMP-9) consists of a prodomain, catalytic domain with 3 fibronectin-like type II modules and C-terminal hemopexin-like (PEX) domain. These domains play distinct roles in terms of proteolytic activity, substrate binding and interaction with inhibitors and receptors. To assess the potential of the MMP-9-PEX domain to interfere with tumor progression, we stably transfected human glioblastoma cells with an expression vector containing a cDNA sequence of the MMP-9-PEX. The selected clones exhibited decreased MMP-9 activity and reduced invasive capacity. We assessed how secretion of MMP-9-PEX by glioblastoma cells affects angiogenic capabilities of human microvascular endothelial cells (HMECs) in vitro. MMP-9-PEX conditioned medium treatment caused a reduction in migration of HMECs and inhibited capillary-like structure formation in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level. The suppression of HMECs survival by conditioned medium from MMP-9-PEX stable transfectants was associated with apoptosis induction characterized by an increase in cells with a sub-G0/G1 content, fragmentation of DNA, caspase-3, -8 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage. A significant tumor growth inhibition was observed in intracranial implants of MMP-9-PEX stable transfectants in nude mice with attenuation of CD31 and MMP-9 protein expression. These results demonstrate that MMP-9-PEX inhibits angiogenic features of endothelial cells and retards intracranial glioblastoma growth. © 2008 Wiley-Liss, Inc. [source] Transgenic mice exhibiting oligodendrocyte-specific expression of a mutant protein tyrosine phosphatase epsilonJOURNAL OF NEUROCHEMISTRY, Issue 2002N. Muja Reversible tyrosine phosphorylation is integral to oligodendrocyte differentiation, and one participant of the phosphorylation cycle, PTP,, is induced in developing oligodendrocytes (Ranjan and Hudson, 1996). To define the role of PTP,, we generated mice expressing a catalytically inactive, hemagglutinin-epitope tagged PTP, (HA-PTP ,) from the 2,,3,-cyclic nucleotide 3,-phosphodiesterase (CNP) promoter. By competing with endogenous, normal PTP, for substrate binding, HA-PTP, would behave in a dominant negative fashion when overexpressed in these mice. Transgene mRNA peaks at postnatal day 21, coincident with the maximal expression of myelin protein mRNAs. Immunohistochemical analyses using antibodies against the HA epitope tag demonstrate that HA-PTP, is expressed in oligodendrocytes, but not in astrocytes and neurons. HA immunoreactivity was present in all myelinated brain structures including the corpus callosum, anterior commissure, and fornix, as well as in the subcortical, cerebellar, and spinal cord white matter. Gross differences in myelination or oligodendrocyte cell density in these brain regions were not detected using antibodies against CNP, myelin basic protein, and an oligodendrocyte marker, CC1. However, by EM axons of the optic nerve appear smaller and less extensively myelinated in transgenic mice than in wild-type littermates. Studies are underway to determine the functional effects of transgene expression on conduction velocity, on the profile of expressed genes, and on potential phosphorylated protein targets of PTP,. [source] Abundant Tissue Butyrylcholinesterase and Its Possible Function in the Acetylcholinesterase Knockout MouseJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Bin Li Abstract: We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well. [source] Functional aspects of ribosomal architecture: symmetry, chirality and regulationJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 11 2004Raz Zarivach Abstract High-resolution structures of both ribosomal subunits revealed that most stages of protein biosynthesis, including decoding of genetic information, are navigated and controlled by the elaborate ribosomal architectural-design. Remote interactions govern accurate substrate alignment within a flexible active-site pocket [peptidyl transferase center (PTC)], and spatial considerations, due mainly to a universal mobile nucleotide, U2585, ensure proper chirality by interfering with D -amino-acids incorporation. tRNA translocation involves two correlated motions: overall mRNA/tRNA (messenger and transfer RNA) shift, and a rotation of the tRNA single-stranded aminoacylated-3, end around the bond connecting it with the tRNA helical-regions. This bond coincides with an axis passing through a sizable symmetry-related region, identified around the PTC in all large-subunit crystal structures. Propelled by a bulged universal nucleotide, A2602, positioned at the two-fold symmetry axis, and guided by a ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry optimal for peptide-bond formation and in geometry ensuring nascent proteins entrance into their exit tunnel. Hence, confirming that ribosomes contribute positional rather than chemical catalysis, and that peptide bond formation is concurrent with A- to P-site tRNA passage. Connecting between the PTC, the decoding center, the tRNA entrance and exit points, the symmetry-related region can transfer intra-ribosomal signals between remote functional locations, guaranteeing smooth processivity of amino acids polymerization. Ribosomal proteins are involved in accurate substrate placement (L16), discrimination and signal transmission (L22) and protein biosynthesis regulation (CTC). Residing on the exit tunnel walls near its entrance, and stretching to its opening, protein-L22 can mediate ribosome response to cellular regulatory signals, since it can swing across the tunnel, causing gating and elongation arrest. Each of the protein CTC domains has a defined task. The N -terminal domain stabilizes the intersubunit-bridge confining the A-site-tRNA entrance. The middle domain protects the bridge conformation at elevated temperatures. The C -terminal domain can undergo substantial conformational rearrangements upon substrate binding, indicating CTC participation in biosynthesis-control under stressful conditions. Copyright © 2004 John Wiley & Sons, Ltd. [source] Exploring functional roles of multibinding protein interfacesPROTEIN SCIENCE, Issue 8 2009Manoj Tyagi Abstract Cellular processes are highly interconnected and many proteins are shared in different pathways. Some of these shared proteins or protein families may interact with diverse partners using the same interface regions; such multibinding proteins are the subject of our study. The main goal of our study is to attempt to decipher the mechanisms of specific molecular recognition of multiple diverse partners by promiscuous protein regions. To address this, we attempt to analyze the physicochemical properties of multibinding interfaces and highlight the major mechanisms of functional switches realized through multibinding. We find that only 5% of protein families in the structure database have multibinding interfaces, and multibinding interfaces do not show any higher sequence conservation compared with the background interface sites. We highlight several important functional mechanisms utilized by multibinding families. (a) Overlap between different functional pathways can be prevented by the switches involving nearby residues of the same interfacial region. (b) Interfaces can be reused in pathways where the substrate should be passed from one protein to another sequentially. (c) The same protein family can develop different specificities toward different binding partners reusing the same interface; and finally, (d) inhibitors can attach to substrate binding sites as substrate mimicry and thereby prevent substrate binding. [source] The oligomeric state and stability of the mannitol transporter, EnzymeIImtl, from Escherichia coli: A fluorescence correlation spectroscopy studyPROTEIN SCIENCE, Issue 8 2006Gertjan Veldhuis Abstract Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeIImtl, is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EIImtl functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EIImtl using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EIImtl is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect. [source] Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiCPROTEIN SCIENCE, Issue 6 2006Erik J. Miller Abstract The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin. [source] Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate bindingPROTEIN SCIENCE, Issue 4 2006Masayo Kotaka Abstract Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylate kinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix ,3 shifting position initially, followed by movement of ,2 to close the substrate site. In addition, we observe significant conformational differences in the TMP-binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to the thymine ring, whereas a cis -proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species. [source] The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesisPROTEIN SCIENCE, Issue 2 2002Ingrid Dreveny Abstract The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R -mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction,the enantiospecific formation of ,-hydroxynitriles,is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C,C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident. [source] The 1.9 Å crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesisPROTEIN SCIENCE, Issue 6 2000Sha Ha Abstract The 1.9 Å X-ray structure of a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis is reported. This enzyme, MurG, contains two ,/, open sheet domains separated by a deep cleft. Structural analysis suggests that the C-terminal domain contains the UDP-GlcNAc binding site while the N-terminal domain contains the acceptor binding site and likely membrane association site. Combined with sequence data from other MurG homologs, this structure provides insight into the residues that are important in substrate binding and catalysis. We have also noted that a conserved region found in many UDP-sugar transferases maps to a ,/,/,/, supersecondary structural motif in the donor binding region of MurG, an observation that may be helpful in glycosyltransferase structure prediction. The identification of a conserved structural motif involved in donor binding in different UDP-sugar transferases also suggests that it may be possible to identify,and perhaps alter,the residues that help determine donor specificity. [source] Structures of apo and GTP-bound molybdenum cofactor biosynthesis protein MoaC from Thermus thermophilus HB8ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Shankar Prasad Kanaujia The first step in the molybdenum cofactor (Moco) biosynthesis pathway involves the conversion of guanosine triphosphate (GTP) to precursor Z by two proteins (MoaA and MoaC). MoaA belongs to the S -adenosylmethionine-dependent radical enzyme superfamily and is believed to generate protein and/or substrate radicals by reductive cleavage of S -adenosylmethionine using an Fe,S cluster. MoaC has been suggested to catalyze the release of pyrophosphate and the formation of the cyclic phosphate of precursor Z. However, structural evidence showing the binding of a substrate-like molecule to MoaC is not available. Here, apo and GTP-bound crystal structures of MoaC from Thermus thermophilus HB8 are reported. Furthermore, isothermal titration calorimetry experiments have been carried out in order to obtain thermodynamic parameters for the protein,ligand interactions. In addition, molecular-dynamics (MD) simulations have been carried out on the protein,ligand complex of known structure and on models of relevant complexes for which X-ray structures are not available. The biophysical, structural and MD results reveal the residues that are involved in substrate binding and help in speculating upon a possible mechanism. [source] Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactionsACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010Radhika Malik The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2,Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+ and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH. [source] Structures of the PKC-, kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533,551 in the C-terminal tail and their roles in ATP bindingACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Tetsuo Takimura Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-,, PKC-, and PKC-,II kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC,substrate complex has been determined. In the previously determined PKC-, complex, residues 533,551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-, kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0,Å resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-, are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-,, which is different from the case of the prototypical AGC-branch kinase PKA. [source] Structure and substrate docking of a hydroxy(phenyl)pyruvate reductase from the higher plant Coleus blumei Benth.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Verena Janiak Hydroxy(phenyl)pyruvate reductase [H(P)PR] belongs to the family of d -isomer-specific 2-hydroxyacid dehydrogenases and catalyzes the reduction of hydroxyphenylpyruvates as well as hydroxypyruvate and pyruvate to the corresponding lactates. Other non-aromatic substrates are also accepted. NADPH is the preferred cosubstrate. The crystal structure of the enzyme from Coleus blumei (Lamiaceae) has been determined at 1.47,Å resolution. In addition to the apoenzyme, the structure of a complex with NADP+ was determined at a resolution of 2.2,Å. H(P)PR is a dimer with a molecular mass of 34,113,Da per subunit. The structure is similar to those of other members of the enzyme family and consists of two domains separated by a deep catalytic cleft. To gain insights into substrate binding, several compounds were docked into the cosubstrate complex structure using the program AutoDock. The results show two possible binding modes with similar docking energy. However, only binding mode A provides the necessary environment in the active centre for hydride and proton transfer during reduction, leading to the formation of the (R)-enantiomer of lactate and/or hydroxyphenyllactate. [source] | |||||||||||||||||||||||||||||||