Absence Of Divalent Metal Research Articles

Design and Development of a Chemical Probe for Pseudokinase Ca2+/calmodulin-Dependent Ser/Thr Kinase.

CASK (Ca2+/calmodulin-dependent Ser/Thr kinase) is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as neurexin kinases with roles implicated in neuronal synapses and trafficking. The lack of a canonical DFG motif, which is altered to GFG in CASK, led to the classification as a pseudokinase. However, functional studies revealed that CASK can still phosphorylate substrates in the absence of divalent metals. CASK dysfunction has been linked to many diseases, including colorectal cancer, Parkinson's disease, and X-linked mental retardation, suggesting CASK as a potential drug target. Here, we exploited structure-based design for the development of highly potent and selective CASK inhibitors based on 2,4-diaminopyrimidine-5-carboxamides targeting an unusual pocket created by the GFG motif. The presented inhibitor design offers a more general strategy for the development of pseudokinase ligands that harbor unusual sequence motifs. It also provides a first chemical probe for studying the biological roles of CASK.

Rapid Thermostabilization of Bacillus thuringiensis Serovar Konkukian 97-27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay.

Thermostabilization of an enzyme with complete retention of catalytic efficiency was demonstrated on recombinant 3-dehydroshikimate dehydratase (DHSase or wtAsbF) from Bacillus thuringiensis serovar konkukian 97-27 (hereafter, B.thuringiensis 97-27). The wtAsbF is relatively unstable at 37 °C, in vitro (t1/237 = 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified asbF library (∼2000 variants) was expressed in E.coli harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3,4-dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized. The half-life of Mut1 at 37 °C was >10-fold higher than the wtAsbF (t1/237 = 169 min). Further, the second-order rate constants for both wtAsbF and Mut1 were approximately equal (9.9 × 105 M-1 s-1, 7.8 × 105 M-1 s-1, respectively), thus demonstrating protein thermostability did not come at the expense of enzyme thermophilicity. In addition, in vivo overexpression of Mut1 in E.coli resulted in a ∼60-fold increase in functional enzyme when compared to the wild-type enzyme under the identical expression conditions. Finally, overexpression of the thermostable AsbF resulted in an approximate 80-120% increase in DHB accumulation in the media relative to the wild-type enzyme.

The hammerhead cleavage reaction in monovalent cations.

Recently, Murray et al. (Chem Biol, 1998, 5:587-595) found that the hammerhead ribozyme does not require divalent metal ions for activity if incubated in high (> or =1 M) concentrations of monovalent ions. We further characterized the hammerhead cleavage reaction in the absence of divalent metal. The hammerhead is active in a wide range of monovalent ions, and the rate enhancement in 4 M Li+ is only 20-fold less than that in 10 mM Mg2+. Among the Group I monovalent metals, rate correlates in a log-linear manner with ionic radius. The pH dependence of the reaction is similar in 10 mM Mg2+, 4 M Li+, and 4 M Na+. The exchange-inert metal complex Co(NH3)3+ also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.

Potent inhibitors of human immunodeficiency virus type 1 integrase: identification of a novel four-point pharmacophore and tetracyclines as novel inhibitors.

A four-point pharmacophore was constructed from energy-minimized structures of chicoric acid and dicaffeoylquinic acid. The search of 206,876 structures in the National Cancer Institute 3D database yielded 179 compounds that contain this pharmacophore. Thirty-nine of these compounds were tested in an in vitro assay specific for human immunodeficiency virus type 1 integrase (IN). Each retrieved structure was fit to the pharmacophore, and the conformation that afforded the best fit was identified. Twenty of the 39 compounds tested exhibited IC50 values of < 20 microM. Among the most potent inhibitors, tetracyclines emerged as a new class of inhibitors. Although the parent tetracycline exhibited marginal potency against purified IN, all substituted tetracyclines tested showed 5-100-fold increased potency. Disintegration assays with truncated IN mutants indicated that tetracyclines inhibit the IN catalytic core domain. To investigate whether chelation of divalent metals is implicated in differential potency of tetracyclines, enzyme assays were performed in the presence of both Mn2+ or Mg2+; no significance difference in potency was observed. Rolitetracycline inhibited IN/DNA complex formation in the presence of EDTA, which suggests that inhibition was metal independent. Rolitetracycline reversed DNA binding of IN after the complex was allowed to form before the addition of drug. Selectivity of tetracyclines was also examined in an assay specific for topoisomerase I, and none of the tetracyclines tested induced topoisomerase I-mediated cleavable complex or inhibited camptothecin-induced cleavable complex. Remarkable potency against the IN in the absence of divalent metals and the core enzyme coupled with water solubility makes tetracyclines potential candidates for X-ray crystal structure determination with IN.

Kinetic analysis of a mutational hot spot in the EcoRV restriction endonuclease.

The EcoRV endonuclease contacts the minor groove of DNA through a peptide loop encompassing residues 67-72. This loop adapts to distorted DNA in the specific complex and to regular DNA in the nonspecific complex. Random mutagenesis had previously identified glutamine 69 as the key component of the loop and this study reports on mutants with glutamate (Q69E), lysine (Q69K), or leucine (Q69L) at this position. The mutants bound DNA specifically at the EcoRV recognition site in the presence of Ca2+, in the same manner as wild-type EcoRV. In the absence of divalent metals, Q69K and Q69L showed the same nonspecific binding as native EcoRV while Q69E failed to bind DNA. Glutamate at position 69 presumably repels nonspecific DNA whilst allowing the adaptations to specific DNA. Both Q69E and Q69K had severely impaired DNA cleavage activities, while Q69L had a steady-state k(cat) within an order of magnitude of wild-type EcoRV though its primary product was nicked DNA, in contrast to double strand breaks by wild-type EcoRV. The activity of Q69L required higher concentrations of Mg2+ than the wild-type and showed a sigmoidal dependence upon the Mg2+ concentration, indicating two metal ions per strand scission. Transient kinetics on Q69L gave lower rate constants for phosphodiester hydrolysis than wild-type EcoRV and its reaction also involved a slow conformational change preceding DNA cleavage that had no equivalent with the wild-type. Gln69 in EcoRV thus plays key roles in the adjustments of the protein to varied DNA structures and in the alignment of the catalytic functions for DNA cleavage.

Mg-Dependent Ecto-ATPase Activity inLeishmania tropica

ATPase activity has been located on the external surface ofLeishmania tropica.SinceLeishmaniais known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5′-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5′-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2varying from 0.1 to 10 mmdid not increase the ATPase activity. At 5 mmATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 ± 0.02 mmMgCl2. The apparentKmfor Mg-ATP2−was 0.13 ± 0.01 mmand free Mg2+did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5= 27.5 ± 1.8 μm).

E461H-.beta.-Galactosidase (Escherichia coli): Altered Divalent Metal Specificity and Slow but Reversible Metal Inactivation

beta-galactosidase (Escherichia coli) with a His substituted for Glu-461 retained about 10% of its normal activity in the absence of divalent metals but was inactivated rather than activated by Mg2+, Mn2+, Zn2+, Ni2+, Cu2+, and Co2+. Since Zn2+, Ni2+, Cu2+, and Co2+ do not interact with wild type beta-galactosidase while Mg2+ and Mn2+ activate and Ca2+ binds but has no effect on wild type beta-galactosidase activity, the substituted enzyme has very different divalent metal interactions. A much larger amount of Mg2+ than of the other divalent metal ions was needed to inactivate the substituted enzyme at pH 7 (half-maximal activity was at 12.5 mM Mg2+ while the half-maximal activities with the other metals were at micromolar levels) compared to the amount of Mg2+ needed to activate the wild type enzyme. The inactivation of E461H-beta-galactosidase caused by Mg2+ took about 20 min. Reactivation by removal of the divalent metal took about 60 min. Interaction with Mg2+ was about 10(7)-fold stronger at pH 9 than at pH 7, and inactivation occurred in less than 2 min at higher pH values. "Galactosylation" (k2, cleavage of the glycosidic bond) seemed to be rate-limiting for E461H-beta-galactosidase at pH values above 6 with both o-nitrophenyl beta-D-galactopyranoside and p-nitrophenyl beta-D-galactopyranoside in both the presence and absence of Mg2+. Mg2+ caused decreases (about 50-fold) of the k2 values of E461H-beta-galactosidase (apparent pKa was about 6.8).(ABSTRACT TRUNCATED AT 250 WORDS)

Overexpression, Purification, and Characterization of VanX, a D-, D-Dipeptidase which Is Essential for Vancomycin Resistance in Enterococcus faecium BM4147

Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the Tryptophan Fluorescence and Hydrodynamic Properties of Rat DNA Polymerase β

We have examined the biophysical properties of DNA polymerase β (β-pol) in solution. Time-resolved and steady-state fluorescence were used to investigate the microenvironment of the lone tryptophanyl residue (Trp324), and a combination of sedimentation equilibrium, sedimentation velocity and fluorescence anisotropy decay measurements were used to study the hydrodynamic properties of the enzyme. Trp324 appears to be exposed to water as judged by the tryptophan emission and steady-state and lifetime quenching experiments. The fluorescence is easily quenched by a neutral quencher acrylamide ( k q = 1.59×10 5M -1s -1), and by a negatively charged ionic quencher, I -( k q = 1.60×10 9M -1s -1), but not by a positively charged ionic quencher, Cs + ( k q = 0.2×10 9M -1s -1). The fluorescence lifetime of β-pol is best described by the sum of two exponentials with a longer lifetime component of 8.4 ns and a shorter lifetime component of 1.3 ns. Decay associated spectra (DAS) show emission maxima at 340 nm and at 345 nm for the shorter lifetime and longer lifetime components, respectively, with corresponding centers of gravity at 347 nm and 348 nm. Sedimentation equilibrium experiments show that the enzyme exists as a monomer at the KCl concentrations (>0.05 M) studied in the absence of divalent metals. Zn 2+ causes higher order aggregation, but no such aggregates are seen with Mg 2+ and Mn 2+. In the presence of 1 mM manganese, the average lifetime decreased approximately 10%, from 8.14 ns to 7.38 ns, with a concomitant increase of average rotational correlational time (φ) from 24 ns to 28 ns. The accessibility of the positively charged quencher (Cs +) to tryptophan also decreases approximately 50%, indicating alteration of the tryptophan microenvironment. By contrast, Mg 2+ causes minor changes in fluorescence properties. The hydrodynamic shape of the intact enzyme and its single-stranded (8 kDa) and double-stranded (31 kDa) DNA binding domains were further investigated by sedimentation velocity measurements. The value of S 0 20.W for the intact enzyme is 2.97 S, and the calculated axial ratio is 5.0. In contrast to the 8 kDa domain, which has a less asymmetric shape with an axial ratio of 2.3, the 31 kDa domain shows an elongated structure with an axial ratio of 5.5. These data suggest that the axial ratio of the intact enzyme may be the result of marked bending of the molecule at the flexible hinge region between the two domains.

Crystal structure of the neutral form of fructose-1,6-bisphosphatase complexed with the product fructose 6-phosphate at 2.1-A resolution.

The crystal structure of fructose-1,6-bisphosphatase (EC 3.1.3.11) complexed with the product fructose 6-phosphate (F6P) has been refined at 2.1-A resolution to an R factor of 0.177 with root-mean-square deviations of 0.014 A and 2.9 degrees from the ideal geometries of bond lengths and bond angles, respectively. The secondary structures but not the trace of the unligated enzyme have been slightly revised in the F6P complex at this higher resolution. Helix H4 in the unligated structure has been refined to a helix-like coil, and two very short 3(10) helices have been found, one in H4 and one in H5. F6P at 10 mM concentration in the absence of divalent metals in our study shows major binding at the active site and minor binding at the AMP site. The major site has almost equal full occupancy in the C1 and C2 chains of the crystallographic asymmetric unit, while the minor site shows occupancy only in the C1 chain at about 50%. The electron density in both (2Fo - Fc) and (Fo - Fc) maps calculated by omitting F6P slightly favors the beta anomer of D-F6P over the alpha anomer. Possible functions of the active-site residues are discussed, and candidates are suggested for site-directed mutagenesis.

Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B

Apolipoprotein B-100 (apoB-100) is the major protein constituent of human plasma low-density lipoproteins (LDL). On the basis of its amino acid sequence [Chen, S.-H., Yang, C.-Y., Chen, P.-F., Setzer, D., Tanimura, M., Li, W.-H., Gotto, A. M., Jr., & Chan, L. (1986) J. Biol. Chem. 261, 12918-12921], apo B-100 is one of the largest monomeric proteins known with a calculated molecular weight of 512937. Heparin binds to the LDL surface by interacting with positively charged amino acid residues of apoB-100, forming soluble complexes in the absence of divalent metals and insoluble complexes in their presence. The purpose of this study was to isolate and characterize the heparin-binding domain(s) of apoB-100. Human plasma LDL were fragmented with cyanogen bromide (CNBr). After delipidation and reduction-carboxymethylation, the CNBr peptides were fractionated by sequential chromatography on DEAE-Sephacel, Mono S, and high reactive heparin (HRH) AffiGel-10; HRH was purified by chromatography of crude bovine lung heparin on LDL AffiGel-10. Heparin-binding peptides were further purified by reverse-phase high-performance liquid chromatography. Heparin-binding activity was monitored by a dot-blot assay with 125I-HRH. The amino-terminal sequences of four CNBr heparin-binding peptides (CNBr-I-IV) were determined. CNBr-I-IV correspond to residues 2016-2151, 3109-3240, 3308-3394, and 3570-3719, respectively, of the amino acid sequence of apoB-100. Each CNBr peptide contains a domain(s) of basic amino acid residues which we suggest accounts for their heparin-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

18] Prenyltransferase from human liver

This chapter describes the isolation of prenyltransferase from human liver. Prenyltransferase has been purified to homogeneity from yeast, pig liver, and in crystalline form from chicken liver. The homogeneous human liver enzyme, the first sterol synthesizing enzyme to be purified from a human tissue, has properties distinct from the enzyme from these sources. Human liver prenyltransferase has an absolute requirement for divalent magnesium or manganese ions for activity. Not more than background activity is observed in the absence of divalent metals. This chapter highlights purification process of this enzyme. It consists of six steps namely—crude extract, ammonium sulfate fractionation, batch treatment with carboxymethyl cellulose, DEAE-cellulose column chromatography, butyl-agarose column chromatography, and calcium phosphate column chromatography.

Trapping of transition metal-nucleotide complexes in myosin subfragment 1 by cross-linking thiols; divalent transition metal probes of the active site.

Recent experiments [Wells, J., & Yount, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966] have shown it is possible to trap MgADP and other nucleotides stably at the active site of myosin by cross-linking two thiol groups. A variety of cross-linking reagents including chelation of the two thiols by cobalt (III) phenanthroline or covalent reaction with N,N'-p-phenylenedimaleimide (pPDM) are effective trapping agents. No trapping of nucleotides occurs in the absence of divalent metals. Thus far Mg2+, Mn2+, Co2+, Ni2+, and Ca2+ but not Zn2+ all function to promote trapping of the 1:1 divalent metal-ADP complex and to enhance the rate of ATPase inactivation. Substitution-inert Cr(III) complexes of ADP, ATP, or pyrophosphate that bind very weakly or not at all to the active site are not trapped by cross-linking. While the stability of the trapped divalent metals varies, e.g., t1/2 of 0.5-7 days at 0 degree C, they are stable enough to permit accurate spectral measurements of the Mn2+ and Co2+ trapped complexes. Electron paramagnetic resonance (EPR) measurements of Mn2+ bound to 5'-adenylyl imidodiphosphate or complexed to myosin chymotryptic subfragment 1 indicate that the metal is bound at the active site. Circular dichroism (CD) and visible absorption studies of the Co2+ . ADP trapped complex indicate the metal ion is in an asymmetric octahedral environment. EPR and CD measurements show that the environment of the metal nucleotide is the same whether bound reversibly or stably trapped at the active site.

The RNA injection step of bacteriophage f2 infection.

SUMMARY In this communication we describe the events occurring during the injection step in the infection of Escherichia coli Hfr cells with the RNA bacteriophage f2. The phage RNA is partially injected into the F-pilus of the Hfr cell. This step was found to be necessary for f2 RNA to block the penetration of the DNA phage f1. A cold sensitive mutant of f2 is described. It is able to inject its RNA at 41° but not at 31°. After contact is made between the phage RNA and the F-pilus, the coat protein of the phage desorbs from the F-pilus as an empty shell, leaving the RNA free to enter the cell. In the absence of divalent metals, however, the phage RNA is unable to leave the shell. When the shell desorbs from the F-pilus, the RNA remains inside in a ribonuclease sensitive state. A small fraction of the RNA in cultures depleted of divalent metal ions remains bound to the cell. It is sensitive to ribonuclease and is removed from the cell by treatments which remove F-pili.