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1.
The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s-1 using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (kcat) and have only a 2-fold effect on the Km for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a potent activator that functions minimally by controlling ATP-Mg binding.  相似文献   

2.
Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.  相似文献   

3.
Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.  相似文献   

4.
Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme. At 4 degreesC, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s-1, and for deuridylylation it is 226 +/- 10 s-1 with Glc-1-P and 166 +/- 10 s-1 with Gal-1-P. Inasmuch as the overall turnover number at 4 degreesC is 62 s-1, the covalent intermediate is kinetically competent. The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites. Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-)4 s-1 and 4.2 x 10(-)4 s-1 at 4 and 27 degreesC, respectively. In experiments with uridylyl-Q168R and glucose-1-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-)4 s-1 and 1.68 x 10(-)3 s-1 at 4 and 27 degreesC, respectively. The value of Km for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R. The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R. The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits. The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol-1 in comparison to the wild-type enzyme. These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions. Instead, the main defect is very slow uridylylation and deuridylation.  相似文献   

5.
Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-in-activating proteins (RIPs), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y114F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A1 form of Slt-IA (wild-type-delta). The same general relationships held: relative to wild type-delta, Y114F-delta was attenuated about 7-fold, and Y114S-delta about 300-fold.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+ [T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994) Biochemistry 33, 6301-6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases from Corynebacterium glutamicum and Escherichia coli, which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+ binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+ site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+ in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated epsilon-amino group of Lys 117 in these bacterial enzymes may provide an "internal monovalent cation." Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is > 200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated epsilon-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.  相似文献   

7.
The Syk protein-tyrosine kinase is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. After cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues. We have previously reported that Syk expressed in COS cells is predominantly phosphorylated at both Tyr518 and Tyr519 at its putative autophosphorylation site. In this study, we have examined the role of each of these two residues for the catalytic activity of Syk in vitro and for the Syk-induced phosphorylation of cellular proteins in intact cells. Mutation of either residue had minor effects on the catalytic activity of Syk, and even the double mutant [F518, F519]Syk was about 60% as active as the wild-type enzyme. In intact cells, however, all three mutants consistently failed to induce the extensive tyrosine phosphorylation of cellular proteins typically observed with wild-type Syk. We have recently shown that the doubly phosphorylated Y518/Y519 site is also the site for association of Syk with the SH2 domain of the Lck kinase, which suggests that although phosphates at Y518/Y519 may enhance the catalytic activity of Syk, its interaction with Src family protein-tyrosine kinases is at least equally important for the induction of downstream substrate phosphorylation.  相似文献   

8.
The mammalian ubiquitin conjugating enzyme known as E2-25K catalyzes the synthesis of polyubiquitin chains linked exclusively through K48-G76 isopeptide bonds. The properties of truncated and chimeric forms of E2-25K suggest that the polyubiquitin chain synthesis activity of this E2 depends on specific interactions between its conserved 150-residue core domain and its unique 50-residue tail domain [Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537]. In the present study, we provide strong support for this model by showing that a point mutation in the core domain (S86Y) mimics the effect of deleting the entire tail domain: the ability to form an E2 approximately ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (>/=100-fold reduction in kcat/Km). The properties of E2-25K enzymes carrying the S86Y mutation indicate that this mutation strengthens the interaction between the core and tail domains: both free and ubiquitin-bound forms of S86Y-25K are completely resistant to tryptic cleavage at K164 in the tail domain, whereas wild-type enzyme is rapidly cleaved at this site. Other properties of S86Y-26K suggest that the active site of this mutant enzyme is more occluded than the active site of the wild-type enzyme. (1) Free S86Y-25K is alkylated by iodoacetamide 2-fold more slowly than the wild-type enzyme. (2) In assays of E2 approximately ubiquitin thiol ester formation, S86Y-25K shows a 4-fold reduced affinity for E1. (3) The ubiquitin thiol ester adduct of S86Y-25K undergoes (uncatalyzed) reaction with dithiothreitol 3-fold more slowly than the wild-type thiol ester adduct. One model to accommodate these findings postulates that an enhanced interaction between the core and tail domains, induced by the S86Y mutation, causes a steric blockade at the active site which prevents access of the incoming ubiquitin acceptor to the thiol ester bond. Consistent with this model, the S86Y mutation inhibits ubiquitin transfer to macromolecular acceptors (ubiquitin and polylysine) more strongly than transfer to small-molecule acceptors (free lysine and short peptides). These results suggest that unique residues proximal to E2 active sites may influence specific function by mediating intramolecular interactions.  相似文献   

9.
The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.  相似文献   

10.
Nyk/Mer receptor tyrosine kinase is a new member of the Ufo/Axl tyrosine kinase family and is characterized by its neural cell adhesion molecule-like extracellular domain. By using a vaccinia virus expression system to express a constitutively activated form of Nyk, we identified the major sites of Nyk autophosphorylation in tryptic peptide IY749SGDY753Y754R. Tyr-749, Tyr-753, and Tyr-754 in this peptide lie in the activation loop of the kinase domain. We also studied a series of Nyk mutants in which the three tyrosine residues were replaced individually, in pairs, or all together by phenylalanine. Single mutations of Tyr-749 or Tyr-753 to phenylalanine reduced Nyk kinase activity toward exogenous substrate to 39 or 10% of that of the wild type Nyk, respectively, whereas the Tyr-754 mutant is completely inactive. All of the double and triple Tyr-Phe mutants reduced Nyk kinase activity to a level below the background. Similar results were obtained when Nyk autophosphorylation levels were examined. Our studies suggest that full activity of Nyk/Mer kinase requires phosphorylation of all three tyrosine residues in the kinase domain (Tyr-749, Tyr-753, and Tyr-754) and that Nyk kinase activity is modulated by the level of autophosphorylation in the kinase domain. Given the highly conserved nature of this region among the Ufo/Axl receptor family members, the information presented in this report may provide insight to the biochemical properties of other members of this family.  相似文献   

11.
Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.  相似文献   

12.
Autophosphorylation of type I receptor tyrosine kinases (RTKs) comprises one step in the signaling events mediated by erbB receptors such as p185neu and EGFR. Previous analysis of p185neu has indicated that there are at least five tyrosine autophosphorylation sites, Y882, Y1028, Y1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have specifically analysed the effect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also deleted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that deletion with Y882F (Y882F/APstop). Both in vitro and in vivo transformation assays showed that substitution of tyrosine882 by phenylalanine significantly decreased the transforming potential of activated, oncogenic p185neu, although no significant difference in the total phosphotyrosine levels of the mutant proteins were observed. To analyse mitogenic signaling in response to ligand, the intracellular domains of p185neu and Y882F were fused with the extracellular domain of the EGF receptor. The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were less responsive to EGF stimulation than those expressing EGFR/neu receptors. In vitro kinase assays demonstrated that abolishing the autophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185neu. These studies, taken together with the phenotypic inhibition observed with cells expressing Y882F, suggest that the tyrosine882 residue may be important for p185neu-mediated transformation by affecting the enzymatic kinase function of the p185neu receptor.  相似文献   

13.
Leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4 and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA4 hydrolase has been suggested as a possible catalytic amino acid. To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site-directed mutagenesis. The mutated cDNAs were expressed in Escherichia coli and the resulting recombinant proteins, named [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA4 into leukotriene B4, and the peptidase activity. None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3% that of the wild-type enzyme. The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10-fold increase in Km for leukotriene A4 compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with Vmax values of 600 nmol.mg-1.min-1 and 1000 nmol.mg-1.min-1 for [Y383Q]LTA4 hydrolase and the wild-type enzyme, respectively. No such effect of substrate concentration could be observed on the peptidase activity. As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively. The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA4 hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA4 into LTB4.  相似文献   

14.
Overexpression of the transmembrane protein-tyrosine phosphatase (PTPase) CD45 in nonhematopoietic cells results in decreased signaling through growth factor receptor tyrosine kinases. Consistent with these data, insulin receptor signaling is increased when the CD45-related PTPase LAR is reduced by antisense suppression in a rat hepatoma cell line. To test whether the hematopoietic cell-specific PTPase CD45 functions in a manner similar to LAR by negatively modulating insulin receptor signaling in hematopoietic cells, the insulin-responsive human multiple myeloma cell line U266 was isolated into two subpopulations that differed in CD45 expression. In CD45 nonexpressing (CD45-) cells, insulin receptor autophosphorylation was increased by 3-fold after insulin treatment when compared to CD45 expressing (CD45+) cells. This increase in receptor autophosphorylation was associated with similar increases in insulin-dependent tyrosine kinase activation. These receptor level effects were paralleled by postreceptor responses. Insulin-dependent tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and Shc was 3-fold greater in CD45- cells. In addition, insulin-dependent IRS-1/phosphatidylinositol 3-kinase association and MAP kinase activation in CD45- cells were also 3-fold larger. While expression of CD45 was associated with a decrease in the responsiveness of early insulin receptor signaling, interleukin 6-dependent activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was equivalent between CD45- and CD45+ cells. These observations indicate that CD45 can function as a negative modulator of growth factor receptor tyrosine kinases in addition to its well-established role as an activator of src family tyrosine kinases.  相似文献   

15.
Syk, a nonreceptor protein-tyrosine kinase, is activated by both oxidative and osmotic stress and plays different roles in the transduction of stress signals. In this study, the regulation of oxidative and osmotic stress induced Syk activation was investigated utilizing Syk-negative DT40 cells, expressing various Syk mutants. Phosphorylation of Y518Y519 was demonstrated to be required for both oxidative and osmotic stress induced Syk activation. Syk activation by these two types of stress stimuli was a combination of both autophosphorylation and the activities of additional tyrosine kinases. Oxidative stress induced Syk tyrosine phosphorylation was almost completely attributed to autophosphorylation, whereas other tyrosine kinases were largely responsible for osmotic stress induced Syk tyrosine phosphorylation. Moreover, the Src homology 2 (SH2) domains of Syk differentially regulated Syk activation. Both mSH2(N) Syk and mSH2(C) Syk, in which the phosphotyrosine-dependent binding motif within the SH2 domains contained point mutations, showed a significantly higher activity than that observed in wild-type Syk, following osmotic stress treatment. In comparison, in response to oxidative stress, only mSH2(N) Syk demonstrated a stronger activation than wild-type Syk. Therefore, differential activation and regulation of Syk may give an insight into the distinctive functions of Syk in oxidative and osmotic stress signaling.  相似文献   

16.
Insulin-like growth factor I action has been implicated in the pathogenesis of many different malignancies, including breast cancer. Insulin-like growth factor I receptors (IGF-IRs) are overexpressed in virtually all breast cancer cell lines, in which they are believed to enhance growth and inhibit apoptosis. In this study, the functional activity of IGF-IRs from normal and malignant human breast tissue was assessed. IGF-IR expression was 14-fold higher in malignant breast tissue than in normal breast tissue. IGF-IR autophosphorylation and kinase activity were 2-4-fold higher in purified receptor preparations from malignant breast tissue as compared to normal breast tissue when normalized for receptor number. This increase in receptor function, coupled with the enhanced receptor expression, amounts to a 40-fold elevation in IGF-IR tyrosine kinase activity in malignant breast tissue. The enhanced receptor autophosphorylation and kinase activity were observed in the absence of hormonal stimulation and seem to result from an alteration in the intrinsic activity of the receptor itself. Protein tyrosine phosphatase activity is also increased in malignant breast tissue. These data suggest that the IGF-IR is an important target for breast cancer therapy.  相似文献   

17.
The biphasic kinetics for phosphorylation of poly(E4Y) by the protein tyrosine kinase pp60c-src were examined. At pH 6.5 substrate inhibition was observed, whereas at pH 8.0 the kinetics were still biphasic, but the enzyme was no longer inhibited. The reaction rate increased in a nonlinear fashion with increasing concentration of substrate. The kinetics were examined from the view that the biphasic kinetics at pH 8.0 were due to two enzymes acting simultaneously on the same substrate. A 55-fold difference in Km values (0.029 versus 1.6 mg/ml) was calculated. The low Km form of the enzyme (0.043 mg/ml) was physically separated from the mixture of kinetic variants by immunoaffinity chromatography, and phosphorylation by protein kinase A resulted in the formation of an enzyme with an intermediate Km (0.3-0.4 mg/ml). The presence of multiple kinetic forms of this tyrosine kinase has important implications in our efforts to understand the role of pp60c-src in human oncology.  相似文献   

18.
Glu-198 of human matrilysin is a conserved residue in the matrix metalloproteinases and is considered to play an important role in catalysis by acting as a general base catalyst toward the zinc-bound water molecule, on the basis of mechanistic proposals for other zinc proteases. In the present study, Glu-198 is mutated into Asp, Cys, Gln, and Ala, and the zinc binding properties, kinetic parameters, and pH dependence of each mutant are determined in order to examine the role of Glu-198 in catalysis. The mutations chosen either modify (C and D) or eliminate (A and Q) the general base properties of residue-198. All the mutants bind 2 mol of zinc per mol of enzyme, indicating that Glu-198 is not crucial to the binding of the catalytic zinc to the enzyme. The value of kcat/Km for the E198D mutant is only 4-fold lower than that of wild-type enzyme at the pH optimum of 7.5, while that for the E198C mutant is decreased by 160-fold. The E198Q and E198A enzymes containing the mutations that have eliminated the nucleophilic and acid/base properties of the residue are still active, having lower kcat/Km values of 590- and 1900-fold, respectively. The decrease in activity of all the mutants is essentially due to a decrease in kcat. The kcat/Km values of the mutants as a function of pH display broad bell-shaped curves that are similar to the wild-type enzyme. The acidic pKa value is not greatly affected by the change in the chemical properties of residue-198. The similarity in the pH profiles for the mutant and wild-type enzymes indicates that the ionization of Glu-198 is not responsible for the acidic pKa. Ionization of the zinc-bound water may be responsible for this pKa since the three His ligands and the scaffolding of the matrilysin catalytic zinc site are different from that observed in carboxypeptidase A and would predict a lower pKa for the metal-bound water. If the zinc-bound water is the nucleophile in the reaction, the role of Glu-198 in catalysis may be to stabilize the transition state or act as a general acid catalyst after the rate-determining step.  相似文献   

19.
Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.  相似文献   

20.
Pre-steady-state kinetic analyses of the catalytic subunit of cAMP-dependent protein kinase showed that the rate constant for phosphoryl transfer is fast and either the release of one or both of the products or a conformational change controls turnover [Grant, B., & Adams, J. A. (1996) Biochemistry 35, 2022-2029]. To determine which step or steps control turnover in the wild-type enzyme, we used a catalytic trapping technique to measure directly the dissociation rate constant for ADP. The phosphorylation of two peptide substrates, LRRASLG and GRTGRRNSI, was monitored using a rapid quench flow technique under conditions where saturating concentrations of ADP were preequilibrated with the enzyme before excess ATP and one of the substrates were added to trap the free enzyme and to start the phosphorylation reaction. Under ADP preequilibration conditions, no 'burst' phase was observed, and although the rate of linear, steady-state turnover was unaffected, the net production of phosphopeptide lagged behind the non-preequilibrated control. This phenomenon occurs due to the slow release of the product, and kinetic modeling suggests that this effect can be explained if the dissociation rate constant for ADP is 24 s-1 and solely limits turnover (kcat = 23 s-1) for the phosphorylation of LRRASLG. Using GRTGRRNSI, the dissociation rate constant for ADP is 35 s-1 and limits turnover (kcat = 29 s-1) if the reaction is initiated by the addition of enzyme. Under preequilibration conditions with either ATP or GRTGRRNSI, turnover is approximately 50% lower, suggesting that ADP release may partially control this parameter. This preequilibration effect can be explained by slowly interconverting enzyme forms with specific peptide-induced turnover properties. These studies indicate that ADP release is an essential rate-limiting component for turnover but also suggests that other factors contribute subtly when the structure of the substrate is altered.  相似文献   

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