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1.
A new phosphoglycerate kinase over-expression vector, pYE-PGK,has been constructed which greatly facilitates the insertionand removal of mutant enzyme genes by cleavage at newly introducedBamtHI sites. This vector has been used to prepare mutant proteinin appreciable (100 mg) quantities for use in kinetic, crystaUographicand NMR experiments. Aspartate 372 is an invariant amino acidresidue in genes known to code for a functionally active PGK.The function of this acidic residue appears to be to help desolvatethe magnesium ion compfexed with either ADP or ATP when thissubstrate binds to the enzyme. Both crystallographk and nuclearmagnetic resonance experiments show that the replacement ofthe residue with asparagine has only minimal effects on theoverall structure. The substitution of the charged carboxylgroup with that of the neutral amide affects the binding ofthe nucleotide substrate as predicted but not, as might havebeen expected, the binding of 3-phospho-glycerate. The overallvelocity of the enzymic reaction (Vmax) is reduced 10-fold bythe substitution of aspartic acid 372 by an asparagine residue(D372N). This reduction in Vmax is considerably less than onewould expect from its known position within the structure ofthe enzyme. This result therefore poses questions about ourunderstanding of charged groups at the active centres of enzymesand of the reason for their apparent conservation.  相似文献   

2.
Residue 31 of porcine pancreatic phospholipase A2 (PLA2) islocated at the entrance to the active site. To study the roleof residue 31 in PLA2, six mutant enzymes were produced by site-directedmutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Seror Gly. Direct binding studies indicated a three to six timesgreater affinity of the Trp31 PLA2 for both monomeric and micellarsubstrate analogs, relative to the wild-type enzyme. The otherfive mutants possess an unchanged affinity for monomers of theproduct analog n-decylphosphocholine and for micelles of thediacyl substrate analog rac-l,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine.The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholinewere decreased 9–20 times for these five mutants. Kineticstudies with monomeric substrates showed that the mutants haveVmax values which range between 15 and 70% relative to the wild-typeenzyme. The Vmax values for micelles of the zwitterionic substratel,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3–50times. The Km values for the monomeric substrate and the kmvalues for the micellar substrate were hardly affected in thecase of five of the six mutants, but were considerably decreasedwhen Trp was present at position 31. The results of these investigationspoint to a versatile role for the residue at position 31: involvementin the binding and orientating of monomeric substrate (analogs),involvement in the binding of the enzyme to micellar substrateanalogs and possibly involvement in shielding the active sitefrom excess water.  相似文献   

3.
Properties of a single-chain antibody containing different linker peptides   总被引:1,自引:0,他引:1  
Single-chain antibodies were constructed using six differentlinker peptides to join the VH and VL domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)3 linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcoreTM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.  相似文献   

4.
The function of aspartic acid residue 101 in the active siteof Escherichia coli alkaline phosphatase was investigated bysite-specific mutagenesis. A mutant version of alkaline phosphatasewas constructed with alanine in place of aspartic acid at position101. When kinetic measurements are carried out in the presenceof a phosphate acceptor, 1.0 M Tris, pH 8.0, both the kcat andthe Km, for the mutant enzyme increase by –2-fold, resultingin almost no change in the kcat/Km ratio. Under conditions ofno external phosphate acceptor and pH 8.0, both the kcat andthe Km for the mutant enzyme decrease by {small tilde}2-fold,again resulting in almost no change in the kcat/Km ratio. Thekcat for the hydrolysis of 4-methyl-umbelliferyl phosphate andp-nitrophenyl phosphate are nearly identical for both the wild-typeand mutant enzymes, as is the K1 for inorganic phosphate. Thereplacement of aspartic acid 101 by alanine does have a significanteffect on the activity of the enzyme as a function of pH, especiallyin the presence of a phosphate acceptor. At pH 9.4 the mutantenzyme exhibits 3-fold higher activity than the wild-type. Themutant enzyme also exhibits a substantial decrease in thermalstability: it is half inactivated by treatment at 49°C for15 min compared to 71°C for the wild-type enzyme. The datareported here suggest that this amino acid substitution altersthe rates of steps after the formation of the phospho-enzymeintermediate. Analysis of the X-ray structure of the wild-typeenzyme indicates that the increase in catalytic rate of themutant enzyme in the presence of a phosphate acceptor may bedue to an increase in accessibility of the active site nearSerl02. The increased catalytic rate of this mutant enzyme maybe utilized to improve diagnostic tests that require alkalinephosphatase, and the reduced heat stability of the mutant enzymemay make it useful in recombinant DNA techniques that requirethe ability to heat-inactivate the enzyme after use.  相似文献   

5.
Platelet-activating factor acetylhydrolases (PAF-AHs) are uniquePLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipidswith a marked preference for very short acyl chains, typicallyacetyl. The recent solution of the crystal structure of the1 catalytic subunit of isoform Ib of bovine brain intracellularPAF-AH at 1.7 Å resolution paved the way for a detailedexamination of the molecular basis of substrate specificityin this enzyme. The crystal structure suggests that the sidechains of Thr103, Leu48 and Leu194 are involved in substraterecognition. Three single site mutants (L48A, T103S and L194A)were overexpressed and their structures were solved to 2.3 Åresolution or better by X-ray diffraction methods. Enzyme kineticsshowed that, compared with wild-type protein, all three mutantshave higher relative activity against phospholipids with sn-2acyl chains longer than an acetyl. However, for each of themutants we observed an unexpected and substantial reductionin the Vmax of the reaction. These results are consistent withthe model in which residues Leu48, Thr103 and Leu194 indeedcontribute to substrate specificity and in addition suggestthat the integrity of the specificity pocket is critical forthe expression of full catalytic function, thus conferring veryhigh substrate selectivity on the enzyme.  相似文献   

6.
Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'1, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcatm)FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below.  相似文献   

7.
We cloned 17 different PCR fragments encoding VH genes of camel(Camelus dromedarius). These clones were derived from the camelheavy chain immunoglobulins lacking the light chain counterpartof normal immunoglobulins. Insight into the camel VH sequencesand structure may help the development of single domain antibodies.The most remarkable difference in the camel VH, consistent withthe absence of the VL interaction, is the substitution of theconserved Leu45 by an Arg or Cys. Another noteworthy substitutionis the Leu11 to Ser. This amino acid normally interacts withthe CH1 domain, a domain missing in the camel heavy chain immunoglobulins.The nature of these substitutions agrees with the increasedsolubility behavior of an isolated camel VH domain. The VH domainsof the camels are also characterized by a long CDR3, possiblycompensating for the absence of the VL contacts with the antigen.The CDR3 lacks the salt bridge between Arg94 and Asp101. However,the frequent occurrence of additional Cys residues in both theCDR1 and CDR3 might lead to the formation of a second internaldisulfide bridge, thereby stabilizing the CDR structure as inthe DAW antibody. Within CDRs of the camel VH domains we observea broad size distribution and a different amino acid patterncompared with the mouse or human VH. Therefore the camel hypervariableregions might adopt structures which differ substantially fromthe known canonical structures, thereby increasing the repertoireof the camel antigen binding sites within a VH.  相似文献   

8.
In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from VH manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.  相似文献   

9.
The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the VH domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the VH or the VL domain, theoptimized version of the affinity peptide termed ‘Strep-tag’allowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidin–alkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions.  相似文献   

10.
Synthetic oligodeoxynucleotides with single methyl phosphonate(mp) substitutions were used for an analysis of the contributionof phosphate contacts to the recognition of the cleavage siteby the restriction endonuclease EcoRV. Only in the last positionwithin the recognition sequence, is the methyl phosphonate substitutiontolerated by the enzyme. The wild-type enzyme cleaves the SPdiastereomer of the oligodeoxynucleotide GACGATATmpCGTC andthe unmodified sequence with equal rates, whereas the RP diastereomeris cleaved much more slowly. Inspection of the crystal structureof an EcoRV–DNA complex revealed that the non-bridgingoxygen atoms of the phosphodiester bond between the T and Cbases are in hydrogen bonding distance of the hydroxyl groupof the amino acid Thr94. We therefore tried to engineer a variantof EcoRV that would prefer a methyl phosphonate linkage overa normal phosphodiester bond and produced mutants with aminoacid exchanges at position 94. One of them, Thr94Val, showsa dramatically reduced activity towards the unmodified DNA anddoes not accept the Rp diastereomer, but cleaves the SP diastereomerwith the same rate as wild-type EcoRV. Its selectivity, i.e.the ratio of cleavage rates determined for the unmodified andmodified substrates, differs by three orders of magnitude fromthat of the wild-type enzyme.  相似文献   

11.
The DNA sequence of bovine microsomal cytochrome b5 has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome bs in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b5 cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D 1HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b5. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (Tm of 73°C thatis significantly higher than that of ‘tryptic’ ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (Tm 78°C) reflected in the values of Hm, Smand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome bs is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein.  相似文献   

12.
Residue 75 on the flap, a beta hairpin loop that partially coversthe active site cleft, is tyrosine in most members of the asparticproteinase family. Site-directed mutagenesis was carried outto investigate the functional role of this residue in Rhizomucorpusilus pepsin, an aspartic proteinase with high milk-clottingactivity produced by the fungus Rhizomucor pusillus. A set ofmutated enzymes with replacement of the amino acid at position75 by 17 other amino acid residues except for His and Gly wasconstructed and their enzymatic properties were examined. Strongactivity, higher than that of the wild-type enzyme, was foundin the mutant with asparagine (Tyr75Asn), while weak but distinctactivity was observed in Tyr75Phe. All the other mutants showedmarkedly decreased or negligible activity, less than 1/1000of that of the wild-type enzyme. Kinetic analysis of Tyr75Asnusing a chromogenic synthetic oligopeptide as a substrate revealeda marked increase in kcat with slight change in Km, resultingin a 5.6-fold increase in kcat/km. When differential absorptionspectra upon addition of pepstatin, a specific inhibitor foraspartic proteinase, were compared between the wild-type andmutant enzymes, the wild-type enzyme and Tyr75Asn, showing strongactivity, had spectra with absorption maxima at 280, 287 and293 nm, whereas the others, showing decreased or negligibleactivity, had spectra with only two maxima at 282 and 288 nm.This suggests a different mode of the inhibitor binding in thelatter mutants. These observations suggest a crucial role ofthe residue at position 75 in enhancing the catalytic efficiencythrough affecting the mode of substrate-binding in the asparticproteinases.  相似文献   

13.
We used directed evolution to convert Bacillus subtilis subtilisinE into an enzyme functionally equivalent to its thermophilichomolog thermitase from Thermoactinomyces vulgaris. Five generationsof random mutagenesis, recombination and screening created subtilisinE 5-3H5, whose half-life at 83°C (3.5 min) and temperatureoptimum for activity (Topt, 76°C) are identical with thoseof thermitase. The Topt of the evolved enzyme is 17°C higherand its half-life at 65°C is >200 times that of wild-typesubtilisin E. In addition, 5-3H5 is more active towards thehydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-typeat all temperatures from 10 to 90°C. Thermitase differsfrom subtilisin E at 157 amino acid positions. However, onlyeight amino acid substitutions were sufficient to convert subtilisinE into an enzyme equally thermostable. The eight substitutions,which include known stabilizing mutations (N218S, N76D) andalso several not previously reported, are distributed over thesurface of the enzyme. Only two (N218S, N181D) are found inthermitase. Directed evolution provides a powerful tool to unveilmechanisms of thermal adaptation and is an effective and efficientapproach to increasing thermostability without compromisingenzyme activity.  相似文献   

14.
A mutant form of pyruvate kinase in which serine 384 has beenmutated to proline has been engineered in the yeast Saccharomycescerevisiae. Residue 384 is located in a helix in a subunit interfaceof the tetrameric enzyme, and the mutation was anticipated toalter the conformation of the helix and hence destabilize theinterface. Previous results indicate that the mutant favoursthe T quarternary conformation over the R conformation, andthis is confirmed by the results presented here. Addition ofphosphoenol-pyruvate (PEP), ADP and fructose-1,6-bisphosphate(Fru 1,6-P2) singly to the wild-type and mutant enzymes resultsin a significant quenching of tryptophan fluorescence (12–44%),and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum.Fluorescence titration experiments showed that PEP, ADP andFru-1,6-P2 induce conformations which have similar ligand-bindingproperties in the wild-type and mutant enzymes. However, theFru-1,6-P2 induced conformation is demonstrably different fromthose induced by either ADP or PEP. The enzymes differ in theirsusceptibility to trypsin digestion and N-ethylmaleimide inhibition.The thermal stability of the enzyme is unaltered by the mutantion.Far-UV CD spectra show that both enzymes adopt a similar overallsencondary structure in solution. Taken together, the resultssuggest that the Ser384-Pro mutaion causes the enzyme to adopta diffenrent tertiary and/or quaternary structure from the wild-typeenzyme and affects the type and extent of the conformationalchanges induced in the enzyme upon ligand binding. A simplifiedminimal reaction mechanism is proposed in which the R and Tstates differ in both affinity and Kcat. Thus, in terms of themodels of cooperativity and allsoteric interaction, pyruvatekinase is both a K and a V system.  相似文献   

15.
The biological importance of Leu15 of epidermal growth factor(EGF) is suggested by its conservation through evolution, itscritical location in the domain–domain interface of EGFand its close proximity to Arg41, a residue that is crucialfor receptor binding and activation. Mutagenesis of Leu15 ofhuman EGF (hEGF) was employed to examine the role of this residuein the ligand-receptor interaction. The relative receptor affinitiesof the hEGF variants, as determined by radioreceptor competitionassays, varied depending on the amino acid substitution. TheL15F, L15W and L15V hEGF analogues had receptor affinities 45,26 and 18% respectively of wild type hEGF. The L15A and L15Ranalogues displayed receptor affinities of only 2.4 and 1.6%relative to wild type hEGF. No binding of the L15E analoguewas detected. The relative agonist activities, as measured byreceptor tyrosine kinase stimulation assays, generally followeda similar trend. The L15F, L15W and L15V analogues stimulatedthe receptor kinase to a level (Vmax) similar to that for wildtype hEGF. A striking difference was observed between the L15Aand L15R variants; although having similar binding affinities,the L15A mutant activated the receptor to only {small tilde}5%of the wild type Vmax in contrast to 53% for the L15R mutant1H-NMR analysis of the L15R and L15A mutants showed only minorstructural alterations that were not sufficient to account forthe dramatic losses in binding and agonist activities. The resultsindicate that both the size and hydrophobicity of the -branchedaliphatic side chain of Leu15 of hEGF are important in the formationof a catalytically active ligand–receptor complex.  相似文献   

16.
Nine single amino add mutations in the active site of Aspergillusawamori glucoamylase were made by cassette mutagenesis to alterthe pH dependence of the enzyme and to determine possible functionsof the mutated residues. The Glul79-Asp mutation expressed inyeast led to a very large decrease in kcat but to no changein Km, verifying this residue's catalytic function. Aspl76-Gluand Glul80-Asp mutations affected Km a more than kcat, implyingthat Aspl76 and Glul80 are involved in substrate binding orstructural integrity. The Leul77-Asp mutation decreased kcatonly moderately, probably by changing the position of the generalacid catalytic group, and did not affect Km. The Trpl78-Aspmutation greatly decreased kcat while increasing Km, showingthe importance of Trpl78 in the active site. Vall81-Asp andAsnl82-Asp mutations changed kinetk values little, suggestingthat Vall81 and Asnl82 are of minor catalytic and structuralimportance. Finally, insertions of Asp or Gly between residues176 and 177 resulted in almost complete loss of activity, probablycaused by destruction of the active site structure. No largechanges in pH dependence occurred in those mutations where kineticvalues could be determined, in spite of the increase in mostcases of the total negative charge. Increases in activationenergy of maltoheptaose hydrolysis in most of the mutant glucoamylasessuggested cleavage of individual hydrogen bonds in enzyme-substratecomplexes.  相似文献   

17.
The luciferase cDNA from the ‘Genji’ firefly, Luciolacruciata, was mutated with hydroxylamine to isolate mutant lucierases.Some of the isolated mutant enzymes produced different colorsof light, ranging from green to red. Five such mutants, producinggreen (max = 558 nm), yellow-orange (max = 595 nm), orange (max= 607 nm) and red light (max = 609 and 612 nm), were analyzed.The mutations were found to be single amino acid changes, fromVal239 to IIe, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser andHis433 to Tyr respectively.  相似文献   

18.
Using point mutated overproducing strains of E.coli, ribonucleaseT1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp,Tyr45Trp or Trp59Tyr and the corresponding double substitutionsTyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr.Steady state kinetics of the transesterification reaction forthe two dinucleoside monophosphate substrates guanylyl-3', 5'-cytidineand guanylyl-3', 5'-adenosine indicate that the tryptophan canbe introduced in different positions within the ribonucleaseT1 molecule without abolishing enzymatic activity. The Trp59Tyrexchange even enhances catalysis of the cleavage reaction (kcat/Km)relative to the wild type enzyme and similar effects are foundwith single tyrosine to tryptophan substitutions. For the pHdependencies of the guanylyl-3', 5'-cytidine transesterificationreaction of wild type ribonuclease T1 and of the variants, typicallybell-shaped curves are observed with a plateau in the rangepH 4.5–7.0. Their shapes and slopes indicate that theenzymes are comparable in their macroscopic pKa, values. AtpH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-foldhigher transesterification activity for guanylyl-3', 5'-adenosineand a 2-fold increase for guanylyl-3', 5'-cytidine comparedto the wild type enzyme, i.e. this variant catalyses the transesterificationof the substrate guanylyl-3', 5'-adenosine with the same orbetter efficiency as guanylyl-3', 5'-cytidine.  相似文献   

19.
The C backbones of the glucose isomerase molecules of Streptomycesrubiginosus and Arthrobacter have been determined by X-ray crystallographyand compared. Each molecule is a tetramer of eight-stranded/ß barrels, and the mode of association of the tetramersis identical in each case. The Arthrobacter electron densityshows four additional amino acids at the carboxyl terminus.There is also an insertion of six amino acids at position 277,and two individual insertions at about positions 348 and 357(numbering according to the Streptomyces structure). There isa close structural homology throughout the whole molecule, whichis most accurate up to position 325. The r.m.s. displacementfor 315 homologous C positions up to this position is 0.92 Å.  相似文献   

20.
The aspartate and tyrosine aminotransferases from Escherichiacoli have 43% sequence identity and nearly identical activesites. Both are equally good enzymes for dicarboxylate substrates,but the latter transaminates aromatic amino acids 1000 timesfaster. In an attempt to discover the critical residues forthis differential substrate specificity, the aspartate aminotransferasemutant V39L has recently been prepared. It showed improved kcal/Kmvalues for aspartate, glutamate and tyrosine and the correspondingoxo acids, mainly due to two to ten times lower Km values. Forexample, the Km values of V39L (wild type) for Asp and Glu are0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallizedwith 30 mM maleate from both polyethylene glycol and ammoniumsulfate. Both structures were solved and refined to R-factorsof 0.22 and 0.20 at 2.85 and 2.5 Å resolution respectively.They bear strong resemblance to the closed structure of thewild type enzyme complexed with maleate. The unexpected featureis that, for the first time, the closed form was produced incrystals grown from ammonium sulfate. It is concluded that themutation has shifted the conformational equilibrium towardsthe closed form, which leads to generally reduced substrateKmS  相似文献   

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