Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change

The strictly conserved active site residue, Asp222, which formsa hydrogen-bonded salt bridge with the pyridine nitrogen atomof the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase(AATase), was replaced with alanine (D222A) in the Escherichiacoli enzyme. The D222A mutant exhibits non–hyberbolicsaturation behavior with amino acid substrates which appearas apparent negative eooperativity in steady–state kineticanalyses. Single turnover progress curves for D222A are welldescribed by the sum of two exponentials, contrasting with themonophasic kinetics of the wild-type enzyme. An active/inactiveheterodimer containing the D222A mutation retains this biphasickinetic response, proving that the observed eooperativity isnot the result of induced allostery. The anomalous behavioris explained by a hysteretic kinetic model involving two slowlyinterconverting enzyme forms, only one of which is catalyticallycompetent. The slow functional transition between the two formshas a half–life of 10 mins. Preincubation of the mutantwith the dicarboxylk inhibitor maleate shifts the equilibriumpopulation of the enzyme towards the catalytically active form,suggesting that the slow transition is related to the domainclosure known to occur upon association of this inhibitor withthe wild-type enzyme. The importance of Asp222 in the chemicalsteps of transamination is confirmed by the l0⁵fold decreasein catalytic competence in the D222A mutant, and by the largeprimary C–deuterium kinetic isotope effect (6.7 versus2.2 for the wild–type). The transamination activity ofthe D222A mutant is enhanced 4– to 20–fold by reconstltutionwith the co-factor analog N–methylpyridoxal–5–phosphate(N–MePLP), and the C–proton abstraction step isless rate determining, as evidenced by the decrease in the primarykinetic isotope effect from 6.7 to 2.3. These results suggestthat the conserved interaction between the protonated pyridinenitrogen of PLP and the negatively charged carboxylate of Asp222is important not only for efficient C–proton abstraction,but also for conformational transitions concomitant with thetransamination process     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Alcohol-induced changes of {beta}-lactoglobulin - retinol-binding stoichiometry

It has been demonstrated using CD that ethanol induces importantsecondary structure changes of ß-lactoglobulin. CDspectra indicate that ß-lactoglobulin secondary structure,which is mainly composed of ß-strands, becomes mostly-helical under the influence of the solvent polarity changes.The midpoint of ß-strand/-helix transition in ß-lactoglobulinis observed at dielectric constant {small tilde}60 (35% ethanol;v/v). According to CD measurements, the ethanol-dependent secondarystructure changes are reversible. The alkylation of lysines-NH₂ in ß-lactoglobulin weakens the central ß-barrelstructure, since the ß-strand/-helix transition midpointof alkylated ß-lactoglobulin is shifted to lower ethanolconcentration (25% ethanol; v/v). ß-Lactoglobulinstructural changes are triggering the dissociation of the ß-lactoglobulin- retinol complex as judged from complete quenching of its fluorescencein ethanol concentration >30% (v/v). However, in 20% ethanol(v/v), ß-lactoglobulin still retains most of its nativesecondary structure as shown by CD and, in this condition, oneß-lactoglobulin molecule binds an additional secondretinol molecule. This suggests that the highly populated speciesobserved around 20% ethanol (v/v) might represent an intermediatestate able to bind two molecules of retinol.     

Experimental and computational mapping of the binding surface of a crystalline protein

Multiple Solvent Crystal Structures (MSCS) is a crystallographictechnique to identify energetically favorable positions andorientations of small organic molecules on the surface of proteins.We determined the high-resolution crystal structures of thermolysin(TLN), generated from crystals soaked in 50–70% acetone,50–80% acetonitrile and 50 mM phenol. The structures ofthe protein in the aqueous–organic mixtures are essentiallythe same as the native enzyme and a number of solvent interactionsites were identified. The distribution of probe molecules showsclusters in the main specificity pocket of the active site anda buried subsite. Within the active site, we compared the experimentallydetermined solvent positions with predictions from two computationalfunctional group mapping techniques, GRID and Multiple CopySimultaneous Search (MCSS). The experimentally determined smallmolecule positions are consistent with the structures of knownprotein–ligand complexes of TLN.     

The differences in conformation between {alpha}-human atrial natriuretic polypeptide, {alpha}-hANP, and its derivative, Met(O)-{alpha}-hANP, in solution

The differences in conformation between -human atrial natriureticpolypeptide (-hANP) and its inactive analog, Met(O)--hANP, havebeen analyzed by nuclear magnetic resonance spectroscopy. Allproton resonances for both peptides were assigned by means ofthe sequential assignment procedure. The three-dimensional structureof -hANP in solution had previously been determined by distancegeometry calculation using distance constraints derived fromnuclear Overhauser effects (NOEs). Here, the three-dimensionalstructure of Met(O)--hANP was determined. The conformationaldifferences between these two molecules were as follows: threesegments of -hANP, Serl–Cys7, Arg11–Ala17 and Gln18–Tyr28,have some ordered structures. In Met(O)--hANP the Gln18-Tyr28region has a similar conformation, while the remaining two regionsdo not have the ordered structure found in -hANP. It is suggestedthat the conserved conformation of the Gln18–Tyr28 regionis required for binding to the ANP receptor and that the slightbiological activity of Met(O)-a-hANP is due to loss of the orderedstructures evoked in the Serl–Cys7 and Arg11–Ala17regions of -hANP.     

Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin)

Single amino acid substitutions were generated in predictedhydrophilic loop regions of the human tumour necrosis factorbeta (TNF-ß) molecule, and the mutant proteins wereexpressed in Escherichia coli and purified. Mutants with singleamino acid changes at either of two distinct loop regions, atpositions aspartic acid 50 or tyrosine 108, were found to havegreatly reduced receptor binding and cytotoxic activity. Thesetwo regions in TNF-ß correspond to known loop regionswhere mutations also result in loss of biological activity ofTNF–, a related cytokine which shares the same cellularreceptors with TNF-ß. The two distinct loops at positions31-34 and 84-89 in the known three-dimensional structure ofTNF- (equivalent to positions 46–50 and 105–110respectively in TNF-ß), lie on opposite sides of theTNF- monomer. When the TNF-a monomer forms a trimer, the twoloops, each from a different subunit of the trimer, come togetherand lie in a cleft between adjacent subunits. Together, thesefindings suggest that a TNF receptor binds to a cleft betweensubunits via surface loops at amino acid residues 31–34and 84–89 in TNF–, and similarly via surface loopsincluding amino acids aspartic acid 50 and tyrosine 108 in TNF–ß.     

Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1–471 and 509–616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441–471) in the crystal structurewraps around an /-barrel formed by residues 1–440. Thesecond (residues 472–508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466–512 (GA1), 485–512 (GA2) and 466–483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities.     

Inhibition of {beta}S-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Mouse _1–30-horse _31–141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ß^S-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the _1–30 region of the chimericchain, the four sequence differences of the region _17-22 couldinduce perturbations of the side chains at ₁₆, ₂₀ and ₂₃, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse _31–141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into human–horse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O₂ affinity of HbS, but in human–horse chimeric-chain it reduces the O₂ affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe ₁ß₁ interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse_1–30, but lower than mouse _1–30. Gln20() synergisticallycomplements the inhibitory propensity of horse _31–141.However, the inhibitory activity of LM–horse chimeric-chain is still lower than that of mouse–horse chimeric-chain. Therefore, perturbation of multiple contact sites inthe _1–30 region of the mouse–horse chimeric -chainand its linkage with the inhibitory propensity of horse _31–141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease.     

Effects of ethylene glycol on the synthesis of ampicillin using immobilized penicillin G acylase

The effects of organic cosolvents on the synthesis of ampicillin from phenylglycine methyl ester (PGME) and 6‐amino penicillanic acid (6‐APA) using immobilized Bacillus megaterium penicillin G acylase have been examined. Several cosolvents were tested for their influence on the enzyme in terms of enzyme stability and hydrophobicity. Among the cosolvents tested, ethylene glycol was found to increase the yield of ampicillin by 39–50%. The effects of ethylene glycol on the pK_a of PGME, the hydrolysis of ampicillin and PGME, and synthetase/amidase and esterase/amidase ratios were also studied. Experimental data indicated that ethylene glycol inhibited more the hydrolysis of the ampicillin than the hydrolysis of the PGME and the synthetase/amidase ratio varied from 0.2 to 0.88 when the concentration (v/v) of the cosolvent increased from 0 to 40%. The enhancement of the synthesis yield was mainly caused by the reduction in the hydrolysis of acyl donor (PGME) and product (ampicillin) in the water–cosolvent system. © 2003 Society of Chemical Industry     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the {alpha}/{beta}-barred packing)

We present here a spectroscopic structural characterizationof octarellin, a recently reported de novo protein modelledon /ß-barrel proteins [K. Go raj, A.Renard and J.A.Martial(1990) Protein Engng, 3, 259–266]. Infrared and Ramanspectra analyses of octarellin‘s secondary structure revealthe expected percentage of -helices (30%) and a higher ß-sheetcontent (40%) than predicted from the design. When the Ramanspectra obtained with octarellin and native triosephosphateisomerase (a natural /ß-barrel) are compared, similarpercentages of secondary structures are found. Thermal denaturationof octarellin monitored by CD confirms that its secondary structuresare quite stable, whereas its native-like tertiary fold is not.Tyrosine residues, predicted to be partially hidden from solvent,are actually exposed as revealed by Raman and UV absorptionspectra. We conclude that the attempted /ß-barrelconformation in octarellin may be loosely packed. The criteriaused to design octarellin are discussed and improvements suggested.     

Protein modification from mutational analysis of an autologous peptide fragment

In the -complementation of ß-galactosidase an N-terminalpeptide fragment (-peptide) of the wild-type enzyme interactswith a defective ß-galactosidase enzyme to restorecapacity for subunit assembly and activity. We have used previouslya random mutagenesis and screening approach to identify a pentapeptideresidue tract in the -peptide that was highly tolerant of residuesubstitution, with some mutations conferring improved function.This tract is of clear importance for -peptide function butis apparently dispensible in the intact parental enzyme. Toinvestigate this further, we selected tract mutations and placedthem into intact ß-galactosidase, at the correspondingN-terminal position as in the -peptide. We then tested whethersuch specific tract sequences conferred properties to the wholeenzyme which could be predicted from the behaviour of the defectiveenzyme complemented with the corresponding mutant -peptide.This was shown for mutations which positively or negativelyaffected enzyme stability. Additionally, a subset of mutationswhich affected complementation efficiency in vivo were predictedto affect the formation of higher-order structures in the intactprotein, and this was observed experimentally. Mutations whichdecreased peptide complementation dramatically decreased thelevel of formation of multimers in the intact protein and amutation which increased peptide complementation produced markedenhancement of multimer formation in a protein with a preexistingimpairment in higher-order structure formation. Such subtleeffects are difficult to detect directly in the whole proteinby randomization/selection approaches, but in the complementingpeptide the role of the residues within the pentapeptide tractis effectively amplified. Identification of residue tracts exhibitingfunctional tolerance to amino acid substitution in an activepeptide fragment can thus be combined with transferral of potentiallyuseful mutant peptide sequences back into the intact protein.Manipulation of a complementation system in this manner affordsa sensitive approach towards targeted improvement of proteins.     

Co-enzyme specificity of 3-isopropylmalate dehydrogenase from Thermits thermophilus HB8

The co–enzyme specificity of 3–isopropylmalate dehydrogenasefrom an extreme thermophile, Thermus thermophilus HB8, was changedfrom NAD to NADP by site–directed mutagenesis Based onsequence comparison of 3–isopropylmalate dehydrogenasesfrom various organisms with NAD– and NADP–dependentisocitrate dehydrogenases, Ser226, Ser253 and De279 of 3–isopropylmalatedehydrogenase were suggested as determining the co–enzymespecificity. These residues were replaced with the correspondingresidues of NADP–dependent isocitrate dehydrogenases;Arg, Gly and Tyr respectively. The single–mutated enzymes,S226R and I279Y, enhanced the activities towards NADP 10–and 3–fold respectively, whereas S253G reduced the activity.Among the multiple–mutated enzymes, the double–mutatedS226R/I279Y increased the catalytic efficiency against NADP( fold) and shifted the specificity for NAD towards NADP mostsignificantly ( 173–fold).     

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin {alpha} and {beta} chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A)ß chain, conserved in mammalian and avian hemoglobins,is located near the functionally important 1–ß2interface and C-terminal region of the ß chain and isreactive to sulfhydryl reagents. The functional roles of thisresidue are still unclear, although regulation of local bloodflow through allosteric S-nitrosylation of this residue is proposed.To clarify the role of this residue and its functional homologyto F9(88) of the chain, we measured oxygen equilibrium curves,UV-region derivative spectra, Soret-band absorption spectra,the number of titratable -SH groups with p-mercuribenzoate andthe rate of reaction of these groups with 4,4'-dipyridine disulfidefor three recombinant mutant Hbs with single amino acid substitutions:AlaCys at 88 (rHb A88C), CysAla at 93ß (rHb C93ßA)and CysThr at 93ß (rHb C93ßT). These Hbs showedincreased oxygen affinities and impaired allosteric effects.The spectral data indicated that the R to T transition upondeoxygenation was partially restricted in these Hbs. The numberof titratable -SH groups of liganded form was 3.2–3.5for rHb A88C compared with 2.2 for Hb A, whereas those for rHbC93ßA and rHb C93ßT were negligibly small. The reductionof rate of reaction with 4,4'-dipyridine disulfide upon deoxygenationin rHb A88C was smaller than that in Hb A. Our experimentaldata have shown that the residues at 88 and 93ß have definiteroles but they have no functional homology. Structure–functionrelationships in our mutant Hbs are discussed.     

Simultaneous Quantification of Plant Glyceroglycolipids Including Sulfoquinovosyldiacylglycerol by HPLC–ELSD with Binary Gradient Elution

Membrane lipids of photosynthetic organisms consist of glycerophospholipids and glyceroglycolipids. We investigated a method for the simultaneous quantitative analysis of neutral and acidic lipids using HPLC–ELSD, and quantified monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). Ten complex lipid classes were separated with a binary gradient system consisting of chloroform and methanol–acetone–water–acetic acid (30:60:9:1, v/v/v/v) with 0.3% triethylamine (pH 4), and were eluted within 16 min. The contents of SQDG in ten edible plants ranged from 3 to 101 mg/100 g, and were positively correlated to the neutral glyceroglycolipids contents.     

Electrospinnability of poly(butylene succinate): Effects of solvents and organic salt on the fiber size and morphology

Submicrosized and nanosized fibers of polymers can be formed easily by electrospinning techniques. However, bead formation can occur if inappropriate solvent systems are used. In this study, we focused on investigating the effects of solvents and organic salt on the electrospinnability of poly(butylene succinate) (PBS). Electrospun PBS fibers were obtained from single‐solvent systems, that is, systems with chloroform (CF) or dichloromethane, at various concentrations (8–30% w/v). Discrete beads and beaded fibers were still found at every PBS concentration. In this study, the electrospinnability of the PBS solutions in CF were improved by the addition of methanol (MeOH) as a cosolvent and an organic salt [alkyl ammonium ethyl sulfate (AAES)]. The obtained fibers were smooth without any beads, and the diameters were affected by the amount of MeOH and the PBS concentration. The electrospinnability of PBS could be enhanced by the addition of a cosolvent with a high dielectric constant or organic salt (AAES). Moreover, the diameters of the electrospun PBS fibers decreased with increasing AAES concentration. We found that the presence of MeOH (30 vol %) and the addition of AAES caused an increase in the crystallinity of the PBS fibers. Therefore, we concluded that bead‐free ultrafine PBS fibers could be obtained through the addition of the cosolvent and the organic salt. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42716.     

Human aldolase B: liver-specific properties of the isozyme depend on type B isozyme group-specific sequences

A series of chimeric enzymes between two human aldolases A,B or C were constructed to identify the molecular regions responsiblefor isozyme-specific functions. Chimeras constructed betweenaldolases A and B were AB34 (an AB chimera connected at position34), ABA34–306 and ABA212–306 (the ABA chimeras).Those between aldolases B and C are BC243, BC263 and BC306 (theBC chimeras connected at positions as indicated), as well asCB55, CB243, CB263 and CB306 (the CB chimeras connected at positionsas indicated), CBC55–263 (a CBC chimera), and BCB55–193,BCB55–306, BCB79–193 and BCB79–306 (the BCBchimeric enzymes). Through the analysis of the properties ofthese chimeras, it was found that for aldolase B, isozyme Bgroup-specific sequences (IGSs)-l and-4 were required for exertingtype B-specific functions, while the IGSs-2 and -3 enhanced,in collaboration with the IGS-1, the catalytic activity of aldolaseB. In addition, the /ß-barrel and the restricted stretches,which were not specified but occupied two distinct regions spanningthe amino acid positions 108–137 (designated connector1) and 243–306 (designated connector 2), were found tobe indispensable for showing full catalytic activity of aldolaseB.     

Effects of Reaction Variables on Fischer–Tropsch Synthesis with Co-Precipitated K/FeCuAlO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> Catalysts

Abstract  

The effects of reduction temperature and reaction temperature, pressure and space velocity on iron-based K/FeCuAlO _x Fischer–Tropsch catalysts prepared by co-precipitation were investigated. The catalyst reduced at 150 °C deactivated quickly due to an abundance of unreduced iron species. With increasing reduction temperature, the iron oxide’s phase transformed from hematite (α-Fe₂O₃) to magnetite (Fe₃O₄) and finally to metallic iron (α-Fe). The induction period to reach steady-state catalytic activity was reduced at increased reduction temperatures due to in situ reduction by syngas during reaction. CO conversion increased with increasing reaction temperature, and selectivity to C₅+ decreased with increasing reaction pressure and space velocity. At reaction temperatures up to of 300 °C, CO₂ formation by the water–gas shift reaction was linearly correlated with the extent of CO conversion, and CO₂ formation was slightly suppressed at ≥350 °C by a reverse water–gas shift reaction.     

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of {alpha}-helices

The 247–260 and 289–299 -helices of Bacillus subtilisneutral protease have a lysine in their N-terminal turn. Theselysines were replaced by Ser or Asp in order to improve electrostaticinteractions with the -helix dipole. After replacing Lys bySer at positions 249 or 290, the thermostability of the enzymewas increased by 0.3 and 1.0°C, respectively. The Asp249and Asp290 mutants exhibited a stabilization of 0.6 and 1.2°C,respectively. The results show the feasibility of stabilizingenzymes by introducing favourable residues at the end of -helices.     

Ir-based oxide electrodes: oxygen evolution reaction from mixed solvents

The influence of 30% (v/v) organic cosolvent in 1.0 mol dm^–3 HClO₄ on the OER electrode kinetics, surface properties and electrode stability of IrO₂-based electrodes was investigated by cyclic voltammetry and polarization curves. The Tafel coefficients in the presence of cosolvent are explained in terms of the change of the rate-determining step (r.d.s.) of the OER electrode mechanism, coating dissolution and/or cosolvent oxidation. Of the several cosolvents investigated t-BuOH and PC show less effects on the OER and electrode properties making them the best choice for organic eletrosynthesis applications, in contrast to AN, which causes coating dissolution, and DMF and DMSO which show an anticipation of the voltammetric current.