Use of methyl iodide for probing the polarity of the immediate environment of --SH groups in thiolenzymes. Reaction of methyl iodide with thiosubtilisin.
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A new approach is proposed for probing the polarity of the immediate environment of -SH groups in thiolenzymes, based on the alkylation of the -SH group with methyl iodide, a relatively small and non-polar molecule. Rate and activation parameters (delta H*, delta S*) for the reaction of the enzyme are compared to those of glutathione, a simple -SH compound alkylated in aqueous medium. The enzyme and model compound are also reacted with iodoacetamide, a polar counterpart of the non-polar methyl iodide. The above method was applied to thiolsubtilisin, an artificial thiolenzyme. 1. The ratio of the rates of alkylation of thiolsubtilisin and glutathione is about 20 times as high with methyl iodide as with iodoacetamide. 2. delta H* and delta S* for enzyme alkylation, as compared to those for glutathione, are remarkably lower with methyl iodide whereas they are slightly higher with iodoacetamide. 3. delta H* and delta S* for alkylation of thiolsubtilisin with methyl iodide are similar to those found with glutathione in 40% dioxane/water mixture. 4. The activation enthalpy and entropy values for the reaction of thiolsubtilisin with D-2-bromo-n-valeramide are lower than those for glutathione reaction. Consequently, in this respect, D-2-bromo-n-valeramide is similar to methyl iodide rather than to iodoacetamide. It is concluded that the -SH group of thiolsubtilisin is located in an environment less polar than water. The concentration of methyl iodide in this non-polar layer is higher than in the bulk solution, which results in an enhanced reaction rate. (+info)
Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus.
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Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation. (+info)
Identification of three key active site residues in the C-terminal domain of human recombinant folylpoly-gamma-glutamate synthetase by site-directed mutagenesis.
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Three cysteines in human recombinant folylpoly-gamma-glutamate synthetase (FPGS) that were reactive with iodoacetamide were located in peptides that were highly conserved across species; the functions of two of these peptides, located in the C-terminal domain, were studied by site-directed mutagenesis. When cDNAs containing mutations in each conserved ionic residue on these peptides were transfected into AUXB1 cells, which lack endogenous FPGS activity, one mutant (D335A) did not complement the auxotrophy, and another (R377A) allowed only minimal growth. FPGS activity could not be detected in insect cells expressing abundant levels of these two mutant proteins from recombinant baculoviruses nor from a virus encoding an H338A mutant FPGS. Kinetic analysis of the purified proteins demonstrated that each of these three mutants was quite different from the others. The major kinetic change detected for the H338A mutation was a 600-fold increase in the K(m) for glutamic acid. For the D335A mutation, the binding of all three substrates (aminopterin, ATP, and glutamic acid) was affected. For R377A, the K(m) for glutamic acid was increased by 1500-fold, and there was an approximately 20-fold decrease in the k(cat) of the reaction. The binding of the K(+) ion, a known activator of FPGS, was affected by the D335A and H338A mutations. We conclude that these three amino acids participate in the alignment of glutamic acid in the active site and that Arg-377 is also involved in the mechanism of the reaction. (+info)
Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously.
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In the crystal structure of the bovine heart mitochondrial F(1)-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded beta subunits, one with MgAMP-PNP bound to the catalytic site (beta(T)) and the other with MgADP bound (beta(D)) have closed conformations. The empty beta subunit (beta(E)) has an open conformation. In beta(T) and beta(D), the distance between the carboxylate of beta-Asp(315) and the guanidinium of beta-Arg(337) is 3.0-4.0 A. These side chains are at least 10 A apart in beta(E). The alpha(3)(betaD311C/R333C)(3)gamma subcomplex of TF(1) with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl(2) with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[(14)C]acetamide after prolonged treatment with CuCl(2) in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a beta subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F(1) are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening. (+info)
Recognition of protein substrates by protein-disulfide isomerase. A sequence of the b' domain responds to substrate binding.
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Refolding of partially folded mitochondrial malate dehydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI). The addition of a 20-fold molar excess of PDI over denatured protein (0. 1 microM) accelerates the recovery of catalytic activity. PDI fluorescence measurements show that 1 mol of PDI binds 1 mol of denatured mMDH when their concentrations approach 1 microM. The binding of PDI, derivatized with the fluorescence probe iodoacetamide fluorescein, to partially folded mMDH is characterized by a dissociation constant of 0.2 microM. It is shown that the fluorescence probe is covalently attached to a SH residue located in the b' domain. Based on the fluorescence measurements of native and derivatized PDI, it is suggested that recognition of the unfolded substrate involves conformational changes propagated to several domains of PDI. (+info)
Reactivity of the two essential cysteine residues of the periplasmic mercuric ion-binding protein, MerP.
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Reactivities of the two essential cysteine residues in the heavy metal binding motif, MTC(14)AAC(17), of the periplasmic Hg(2+)-binding protein, MerP, have been examined. While Cys-14 and Cys-17 have previously been shown to be Hg(2+)-binding residues, MerP is readily isolated in an inactive Cys-14-Cys-17 disulfide form. In vivo results demonstrated that these cysteine residues are reduced in the periplasm of Hg(2+)-resistant Escherichia coli. Denaturation and redox equilibrium studies revealed that reduced MerP is thermodynamically favored over the oxidized form. The relative stability of reduced MerP appears to be related to the lowered thiol pK(a) (5.5) of the Cys-17 side chain. Despite its much lower pK(a), the Cys-17 thiol is far less accessible than Cys-14, reacting 45 times more slowly with iodoacetamide at pH 7.5. This is reminiscent of proteins such as thioredoxin and DsbA, which contain a similar C-X-X-C motif, except in those cases the more exposed thiol has the lowered pK(a). In terms of MerP function, electrostatic attraction between Hg(2+) and the buried Cys-17 thiolate may be important for triggering the structural change that MerP has been reported to undergo upon Hg(2+) binding. Control of cysteine residue reactivity in heavy metal binding motifs may generally be important in influencing specific metal-binding properties of proteins containing them. (+info)
Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1.
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Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group. (+info)
Proteolysis of heat shock transcription factor is associated with apoptosis in rat Nb2 lymphoma cells.
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Previously, we reported that prolactin (PRL)-dependent Nb2 lymphoma cells exhibit an aberrant heat shock response because of cysteine protease-mediated fragmentation of the heat shock transcription factor (HSF). Moreover, exposure of the cells to PRL abrogated heat-induced HSF proteolysis. The present study was conducted to investigate whether HSF proteolysis is a component of the apoptotic process in this model. Initially, the effect of heat stress (41 degrees C for 1 h) on apoptosis, determined by agarose gel electrophoresis and flow cytometric analysis, was evaluated in PRL-dependent Nb2-11 cells and in an autonomous subline (Nb2-SFJCD1). Heat was found to induce HSF proteolysis concomitant with activation of apoptosis in each cell line; treatment with PRL blocked these effects. To determine whether HSF proteolysis occurred as a generalized phenomenon associated with apoptosis, the effects of other activators of this process were evaluated. Vinblastine, cycloheximide, and thapsigargin stimulated fragmentation of HSF and hydrolysis of DNA in each cell line. The addition of PRL blocked the effects of vinblastine but was ineffective in cells treated with either cycloheximide or thapsigargin. Iodoacetamide, a cysteine protease inhibitor that blocks HSF fragmentation, also inhibited apoptosis. In addition, Z-VAD, a general caspase antagonist, blocked vinblastine-induced fragmentation of HSF and DNA, suggesting that the enzyme responsible for proteolysis of the transcription factor was likely a caspase family member. The results suggest that proteolysis of HSF reflects the action of one or more caspases activated as a consequence of stimulation of cell death. It is concluded that HSF may represent a previously unrecognized substrate for caspases or other cysteine proteases activated during apoptosis. (+info)