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1.
Comparisons of rbcL genes for the large subunit of ribulose-bisphosphate carboxylase from closely related C3 and C4 plant species 总被引:2,自引:0,他引:2
G S Hudson J D Mahon P A Anderson M J Gibbs M R Badger T J Andrews P R Whitfeld 《The Journal of biological chemistry》1990,265(2):808-814
Ribulosebisphosphate carboxylase/oxygenase from C4 plants exhibits higher turnover rates and lower affinities for CO2 than the enzyme from C3 plants or C3-C4 intermediate species. This property is shown to be inherited maternally in reciprocal interspecific crosses between two Flaveria species, and thus must be specified by the chloroplast-encoded large subunits. To investigate the amino acid changes responsible, the chloroplast rbcL genes from three pairs of C3 and C4 species from three genera (Flaveria, Atriplex, and Neurachne) were cloned and sequenced. Comparisons of the predicted amino acid sequences from species of the same genus revealed a limited number of changes within each pair, ranging from three to six, of which only one (309Met (C3) to Ile (C4] was consistently observed. This residue occurs in the loop connecting the carboxyl end of beta strand 5 with the amino end of alpha helix 5 in the alpha/beta barrel of the large subunit, and is close to the active site in a region which makes interdomain and intersubunit contacts. However, it is unlikely that a change of this residue alone is responsible for the alteration of kinetic properties. Nucleotide sequence comparisons of the rbcL genes showed no significant or consistent changes in the promoter and transcribed but nontranslated regions to suggest why rbcL is not expressed in C4 leaf mesophyll cells. It is concluded that mutations in rbcL have led to an alteration of the kinetics but not the expression of ribulose-bisphosphate carboxylase. 相似文献
2.
Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions. 相似文献
3.
Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.
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G. J. Lee K. A. McDonald B. A. McFadden 《Protein science : a publication of the Protein Society》1993,2(7):1147-1154
The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme. 相似文献
4.
Claude D. Stringer I.Lucile Norton Fred C. Hartman 《Archives of biochemistry and biophysics》1981,208(2):495-501
Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [3H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys1Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys1AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys1 ThrThrAsxAsxPheThrArg AlaCys1ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys1HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase. 相似文献
5.
Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects.
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K. Assemat P. M. Alzari J. Clment-Mtral 《Protein science : a publication of the Protein Society》1995,4(12):2510-2516
The internal residue Phe 25 in Rhodobacter sphaeroides thioredoxin was changed to five amino acids (Ala, Val, Leu, Ile, Tyr) by site-directed mutagenesis, and the mutant proteins were characterized in vitro and in vivo using the mutant trxA genes in an Escherichia coli TrxA- background. The substitution F25A severely impaired the functional properties of the enzyme. Strains expressing all other mutations can grow on methionine sulfoxide with growth efficiencies of 45-60% that of the wild type at 37 degrees, and essentially identical at 42 degrees. At both temperatures, however, strains harboring the substitutions F25V and F25Y had lower growth rates and formed smaller colonies. In another in vivo assay, only the wild type and the F25I substitution allowed growth of phage T3/7 at 37 degrees, demonstrating that subtle modifications of the protein interior at position 25 Ile/Leu or Phe/Tyr) can produce significant biological effects. All F25 mutants were good substrates for E. coli thioredoxin reductase. Although turnover rates and apparent Km values were significantly lower for all mutants compared to the wild type, catalytic efficiency of thioredoxin reductase was similar for all substrates. Determination of the free energy of unfolding showed that the aliphatic substitutions (Val, Leu, Ile) significantly destabilized the protein, whereas the F25Y substitution did not affect protein stability. Thus, thermodynamic stability of R. sphaeroides thioredoxin variants is not correlated with the distinct functional effects observed both in vivo and in vitro. 相似文献
6.
An isoleucine residue within the carboxyl-transferase domain of multidomain acetyl-coenzyme A carboxylase is a major determinant of sensitivity to aryloxyphenoxypropionate but not to cyclohexanedione inhibitors 总被引:1,自引:0,他引:1
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A 3,300-bp DNA fragment encoding the carboxyl-transferase domain of the multidomain, chloroplastic acetyl-coenzyme A carboxylase (ACCase) was sequenced in aryloxyphenoxypropionate (APP)-resistant and -sensitive Alopecurus myosuroides (Huds.). No resistant plant contained an Ile-1,781-Leu substitution, previously shown to confer resistance to APPs and cyclohexanediones (CHDs). Instead, an Ile-2,041-Asn substitution was found in resistant plants. Phylogenetic analysis of the sequences revealed that Asn-2,041 ACCase alleles derived from several distinct origins. Allele-specific polymerase chain reaction associated the presence of Asn-2,041 with seedling resistance to APPs but not to CHDs. ACCase enzyme assays confirmed that Asn-2,041 ACCase activity was moderately resistant to CHDs but highly resistant to APPs. Thus, the Ile-2,041-Asn substitution, which is located outside a domain previously shown to control sensitivity to APPs and CHDs in wheat (Triticum aestivum), is a direct cause of resistance to APPs only. In known multidomain ACCases, the position corresponding to the Ile/Asn-2,041 residue in A. myosuroides is occupied by an Ile or a Val residue. In Lolium rigidum (Gaud.), we found Ile-Asn and Ile-Val substitutions. The Ile-Val change did not confer resistance to the APP clodinafop, whereas the Ile-Asn change did. The position and the particular substitution at this position are of importance for sensitivity to APPs. 相似文献
7.
Rubisco, the primary photosynthetic carboxylase, evolved 3-4 billion years ago in an anaerobic, high CO(2) atmosphere. The combined effect of low CO(2) and high O(2) levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO(2) and O(2), leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C(4) photosynthesis largely solves this problem by facilitating a high CO(2)/O(2) ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcat(CO2) and lower CO(2)/O(2) specificity (S(C/O)) is well supported, but the structural basis for the differences between C(3) and C(4) Rubiscos is not clear. Flaveria (Asteraceae) includes C(3), C(3)-C(4) intermediate, and C(4) species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C(3) to C(4) photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C(4) Rubiscos from the ancestral C(3) species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic "C(4)" SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus. 相似文献
8.
The carboxylterminal octapeptide of ribulosebisphosphate carboxylase from Rhodospirillum rubrum, which lacks small subunits, shows homology to a highly conserved region near the amino terminus of the small subunits of hexadecameric ribulosebisphosphate carboxylases, which are composed of large and small subunits. Truncations of the R. rubrum enzyme, which partially or completely deleted the region of homology, demonstrated that the region is not an important determinant of the catalytic efficiency of the enzyme. A further truncation, which replaced the carboxylterminal 19 amino acid residues with a single terminal leucyl residue, yielded a Rubisco whose substrate-saturated catalytic rate resembled that of the wild-type enzyme but which had weaker affinities for ribulose-P2 and CO2. 相似文献
9.
Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase 总被引:2,自引:0,他引:2
Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO2 and Mg2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO2 and Mg2+. Incubation of the carboxylase with potassium [14C] cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioactive carboxylase indicated that the reagent carbamylates both NH2-terminal groups and lysyl residues in the large and small subunits. Comparison of the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO2 binding sites on the enzyme. 相似文献
10.
The hGSTM3 subunit, which is preferentially expressed in germ-line cells, has the greatest sequence divergence among the human mu class glutathione S-transferases. To determine a structural basis for the catalytic differences between hGSTM3-3 and other mu class enzymes, chimeric proteins were designed by modular interchange of the divergent C-terminal domains of hGSTM3 and hGSTM5 subunits. Replacement of 24 residues of the C-terminal segment of either subunit produced chimeric enzymes with catalytic properties that reflected those of the wild-type enzyme from which the C-terminus had been derived. Deletion of the tripeptide C-terminal extension found only in the hGSTM3 subunit had no effect on catalysis. The crystal structure determined for a ligand-free hGSTM3 subunit indicates that an Asn212 residue of the C-terminal domain is near a hydrophobic cluster of side chains formed in part by Ile13, Leu16, Leu114, Ile115, Tyr119, Ile211, and Trp218. Accordingly, a series of point mutations were introduced into the hGSTM3 subunit, and it was indeed determined that a Y119F mutation considerably enhanced the turnover rate of the enzyme for nucleophilic aromatic substitution reactions. A more striking effect was observed for a double mutant (Y119F/N212F) which had a k(cat)/K(m)(CDNB) value of 7.6 x 10(5) s(-)(1) M(-)(1) as compared to 4.9 x 10(3) s(-)(1) M(-)(1) for the wild-type hGSTM3-3 enzyme. The presence of a polar Asn212 in place of a Phe residue found in the cognate position of other mu class glutathione S-transferases, therefore, has a marked influence on catalysis by hGSTM3-3. 相似文献
11.
Helfand MS Hujer AM Sönnichsen FD Bonomo RA 《The Journal of biological chemistry》2002,277(49):47719-47723
Infections with bacteria that contain hydrolytic beta-lactamase enzymes are becoming a serious problem in the United States. Mutations at Met-69, an amino acid proximal to the active site Ser-70 in the TEM-1 and SHV-1 beta-lactamases, have emerged as a puzzling cause of bacterial resistance to inhibitors of beta-lactamases. Site-saturation mutagenesis of the 69 position in SHV beta-lactamase was performed to determine how mutations of this non-catalytic residue play a role in increasing 50% inhibitory concentrations (IC(50) concentrations) for clinically important beta-lactamase enzyme inhibitors. Two distinct phenotypes are evident in the variant beta-lactamases studied: significantly increased minimum inhibitory concentrations (microg/ml) and IC(50) concentrations to clavulanic acid for the Met69Ile, Leu, and Val substitutions, and unanticipated increased minimum inhibitory concentrations and hydrolytic activity toward ceftazidime, an advanced generation cephalosporin antibiotic, for the Met69Lys, Tyr- and Phe-substituted enzymes. Molecular modeling studies emphasize the conserved structure of these substitutions despite great variation in substrate specificity. This study demonstrates the key role of Met-69 in defining substrate specificity of SHV beta-lactamases and alerts us to new phenotypes that may emerge clinically. 相似文献
12.
Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity. 相似文献
13.
Conley A Mapes S Corbin CJ Greger D Graham S 《Molecular endocrinology (Baltimore, Md.)》2002,16(7):1456-1468
The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I(133)M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B'-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I(133)M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile(133) to either alanine (I(133)A) or tyrosine (I(133)Y) decreased apparent enzyme activity, but the I(133)A mutant was sensitive to etomidate inhibition, suggesting that it is Ile(133) that decreases etomidate binding rather than Met(133) increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I(133)M mutant and the native placental isozyme. These data suggest that Ile(133) is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees. 相似文献
14.
K. Purohit Robert R. Becker Harold J. Evans 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,715(2):230-239
Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco. 相似文献
15.
Synthesis and assembly of ribulosebisphosphate carboxylase enzyme during greening of barley plants 总被引:2,自引:0,他引:2
M A Smith R S Criddle L Peterson R C Huffaker 《Archives of biochemistry and biophysics》1974,165(2):494-504
The synthesis and activation of ribulosebisphosphate carboxylase was studied in etiolated barley leaves during increasing periods of light irradiation. Comparisons were made among enzymatic activity, 14C-amino acid incorporation into anti-ribulosebisphosphate carboxylase precipitable and 16S protein, and total mass of enzyme. A major portion of newly synthesized anti-ribulosebisphosphate carboxylase specific protein preceded light-induced increase in enzyme activity by a significant period of time. These findings are consistent with a model in which both subunits of ribulosebisphosphate carboxylase are synthesized in response to an early event in greening and subsequently become associated to active oligomeric carboxylase species. 相似文献
16.
Polyak SW Chapman-Smith A Mulhern TD Cronan JE Wallace JC 《The Journal of biological chemistry》2001,276(5):3037-3045
Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL. 相似文献
17.
Random mutagenesis of Thermus thermophilus 3-isopropylmalate dehydrogenase revealed that a substitution of Val126Met in a hinge region caused a marked increase in specific activity, particularly at low temperatures, although the site is far from the binding residues for 3-isopropylmalate and NAD. To understand the molecular mechanism, residue 126 was substituted with one of eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile or Phe. Circular dichroism analyses revealed a decreased thermal stability of the mutants (Delta T ((1/2))= 0-13 degrees C), indicating structural perturbations caused by steric conflict with surrounding residues having larger side chains. Kinetic parameters, k(cat) and K(m) values for isopropylmalate and NAD, were also affected by the mutation, but the resulting k(cat)/K(m) values were similar to that of the wild-type enzyme, suggesting that the change in the catalytic property is caused by the change in free-energy level of the Michaelis complex state relative to that of the initial state. The kinetic parameters and activation enthalpy change (Delta H (double dagger)) showed good correlation with the van der Waals volume of residue 126. These results suggested that the artificial cold adaptation (enhancement of k(cat) value at low temperatures) resulted from the destabilization of the ternary complex caused by the increase in the volume of the residue at position 126. 相似文献
18.
Ikehara T Ikehara S Imamura S Shinjo F Yasumoto T 《Biochemical and biophysical research communications》2007,354(4):1052-1057
Protein phosphatase 2A (PP2A) is composed of structural (A), catalytic (C), and regulatory (B) subunits. The catalytic subunit (PP2A(C)) undergoes reversible carboxyl-methylation and -demethylation at its C-terminal leucine residue (Leu309), catalyzed by PP2A-methyltransferase (PMT) and PP2A methylesterase (PME-1), respectively. In this study, we observed that the activity of PP2A was largely unaffected by the addition of PME-1, and that the regulatory subunit (PR55/B) could bind demethylated PP2A(D). Furthermore, to study the precise effect of Leu309 demethylation on PP2A activity, we generated two His(8)-tagged mutant versions of PP2A(C) containing an alanine residue in place of Leu309, and a deletion of Leu309. Both recombinant mutants exhibited phosphatase activity. In addition, we demonstrated that both mutants could constitute a holoenzyme with the regulatory A and B subunits. Our collective results indicate that methylation of Leu309 of PP2A(C) is unnecessary for the PP2A activity and the binding of PR55/B. 相似文献
19.
The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 ? resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme. 相似文献
20.
Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins. 总被引:5,自引:4,他引:1
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M. T. Bihoreau V. Baudin M. Marden N. Lacaze B. Bohn J. Kister O. Schaad A. Dumoulin S. J. Edelstein C. Poyart et al. 《Protein science : a publication of the Protein Society》1992,1(1):145-150
Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献