Probing the role of threonine and serine residues of E.coli asparaginase II by site-specific mutagenesis

Site-specific mutagenesis has been used to probe amino acidresidues proposed to be critical in catalysis by Escherichiacoli asparaginase II. Thr12 is conserved in all known asparaginases.The catalytic constant of a T12A mutant towards L-aspartk acidß-hydroxamate was reduced to 0.04% of wild type activity,while its An, and stability against urea denaturation were unchanged.The mutant enzyme T12S exhibited almost normal activity butaltered substrate specificity. Replacement of Thr119 with Alaled to a 90% decrease of activity without markedly affectingsubstrate binding. The mutant enzyme S122A showed normal catalyticfunction but impaired stability in urea solutions. These dataindicate that the hydroxyl group of Thr12 is directly involvedin catalysis, probably by favorably interacting with a transitionstate or intermediate. By contrast, Thr119 and Ser122, bothputative target sites of the inactivator DONV, are functionallyless important.     

Deletion of the four C-terminal residues of PepC converts an aminopeptidase into an oligopeptidase

The aminopeptidase PepC is a cysteine peptidase isolated fromlactic acid bacteria. Its structural and enzymatic propertiesclosely resembles those of the bleomycin hydrolases, a groupof cytoplasmic enzymes isolated from eukaryotes. Previous biochemicaland structural data have shown that the C-terminal end of PepCpartially occupies the active site cleft. In this work the substratespecificity of PepC was engineered by deletion of the four C-terminalresidues. The mutant PepC432–435 cleaved peptide substratesas an oligopeptidase while the aminopeptidase specificity wastotally abolished. The substrate size dependency indicated thatPepC432–435 possesses an extended binding site able toaccommodate four residues of the substrate on both sides ofthe cleaved bond. The activity of PepC432–435 towardstryptic fragments of casein revealed a preference for peptideswith hydrophobic amino acids at positions P2 and P3 and forGly, Asn and Gln at position P1. PepC432–435 was shownto be highly sensitive to the thiol peptidase inhibitors leupeptinor E64 which are inefficient towards the wild-type PepC. Inconclusion, deletion of the four C-terminal residues in PepCproduces a new enzyme with properties resembling those of anendopeptidase from the papain family.     

The role of arginines in stabilizing the active open-lid conformation ofRhizomucor miehei lipase

Molecular dynamics simulations for the lid covering the active site ofRhizomucor miehei lipase [EC 3.1.1.3] postulated that, among other interactions, Arg86 in the lid stabilized the open-lid conformation of the protein by multiple hydrogen bonding to the protein surface. Chemical modification of arginine residues inR. miehei lipase with 1,2-cyclohexanedione or phenylglyoxal resulted in residual activities in the hydrolysis of tributyrin of 66 and 46%, respectively. Tryptic maps of native and phenylglyoxal-reactedR. miehei lipase showed that Arg86 was the residue modified most, when the lipase was inhibited to the greatest extent. Guanidine, a structural analog to an arginine side chain, inhibited both the natibe enzyme and the arginine-modified enzymes, resulting in residual activities of 26% as compared to the native enzyme. The inhibition was not an effect of enzyme denaturation. The native enzyme was also inhibited by 1-ethylguanidine, benzamidine and urea, but to a lesser degree than by guanidine. Lipases fromHumicola lanuginosa and porcine pancreas in 100 mM guanidine showed residual activities of 88 and 70%, respectively. The lipases fromCandida antarctica, C. rugosa, Pseudomonas cepacia andP. fluoresences were not inhibited by guanidine. The inhibition ofR. miehei lipase by structural analogs of the arginine side chain and after chemical modification of arginine residues suggest a role of an arginine residue in stabilizing the active open-lid conformation of the enzyme.     

Assessing the role of tryptophan residues in the binding site

Instead of looking at the interfacial area as a measure of theextent of a protein–protein recognition site, a new procedurehas been developed to identify the importance of a specificresidue, namely tryptophan, in the binding process. Trp residueswhich contribute more towards the free energy of binding havetheir accessible surface area reduced, on complex formation,for both the main-chain and side-chain atoms, whereas for theless important residues the reduction is restricted only tothe aromatic ring of the side chain. The two categories of residuesare also distinguished by the presence or absence of hydrogenbonds involving the Trp residue in the complex. A comparisonof the observed change in the accessible surface area with thevalue calculated using an analytical expression provides anotherway of characterizing the Trp residues critical for bindingand this has been used to identify such residues involved inbinding non-proteinaceous molecules in protein structures.     

Probing the role of aromaticity in the design of dipeptide based nanostructures

Self-assembly of peptide into nanostructures is believed to be stabilized primarily by aromatic interactions. Using a minimalistic approach, we probed the importance of aromatic interactions in the self-assembly of simple model dipeptides. Our results suggest that aromaticity may not be absolutely essential for self-assembly, even though it tends to provide directionality to the assembly. We found that peptides containing cyclic/linear side chain hydrophobic residues were also capable of forming stable self-assemblies that are stabilized by hydrophobic interactions. Our observations will find relevance in the design of small peptide based nanoparticles.     

Probing the catalytically essential residues of the {alpha}-L-arabinofuranosidase from Thermobacillus xylanilyticus

The     

Electropolymerization of m-cresol in the presence of trimethylamine

The feasibility of anodic electropolymerization of metacresol (mC) in the presence of trimethylamine (TMA) was demonstrated on a platinum electrode in acetonitrile (AN), tetrahydrofuran (THF), n, n-dimethyl formamide (DMF) and methanol (MeOH) solutions. In DMF and McOH, due to the adsorption of TMA on the electrode, the electropolymerization occurs in the vicinity of the electrode rather than on the electrode surface. The coatings obtained have poor corrosion inhibition characteristics. But in AN and THF, thin coatings were obtained on the electrode as mC is preferentially adsorbed rather than TMA and, subsequently, the electropolymerization takes place on the electrode surface. The polymers obtained have remarkable corrosion protection and thermal stability. The polymers contain C-O-C linkages and have no troublesome nitrogen content.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase

Site-directed mutagenesis was used to alter active-site residuesof methylamine dehydrogenase (MADH) from Paracoccus denitrificans.Four residues of the ß subunit of MADH which are inclose proximity to the tryptophan tryptophylquinone (TTQ) prostheticgroup were modified. The crystal structure of MADH reveals thateach of these residues participates in hydrogen bonding interactionswith other active-site residues, TTQ or water. Relatively conservativemutations which removed the potentially reactive oxygens onthe side chains of Thr122, Tyr119, Asp76 and Asp32 each resultedin greatly reduced or undetectable levels of MADH production.The reduction of MADH levels was determined by assays of activityand Western blots of crude extracts with antisera specific forthe MADH ß subunit. No activity or cross-reactiveprotein was detected in extracts of cells expressing D76N, T122Aand T122C MADH mutants. Very low levels of active MADH wereproduced by cells expressing D32N, Y119F, Y119E and Y119K MADHmutants. The Y119F and D32N mutants were purified from cellextracts and found to be significantly less stable than wild-typeMADH. Only the T122S MADH mutant was produced at near wild-typelevels. Possible roles for these amino acid residues in stabilizingunusual structural features of the MADH ß subunit,protein folding and TTQ biosynthesis are discussed.     

Modeling the role of stabilizing additives during melt recycling of high‐density polyethylene

Post‐use high‐density polyethylene, almost devoid of any stabilizing agents, was restabilized in various degrees and subjected to multiple extrusion cycles at different reprocessing temperatures for assessing its chemical stability. The process‐induced material degradation was attributed primarily to long‐chain branching caused by crosslinking. It was monitored by an increase in viscosity and evaluated on the basis of an approximate expression derived using fundamental principles of macromolecular rheology. It was determined that long‐chain branching increases with temperature and the extent of processing, while decreasing with the amount of restabilizing agent added. A simple model was developed to quantitatively describe the progress of the chemical change by relating it to key material and operational variables. Besides constituting a useful method of monitoring and controlling polymer modification during processing, this model suggests ways of optimizing stabilization according to the particular processing and product requirements. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 2207–2217, 2001     

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin

Chemically prepared hevein domains (HDs), N-terminal domainof an antifungal protein from Nicotiana tabacum (CBP20-N) andan antimicrobial peptide from Amaranthus caudatus (Ac-AMP2),were examined for their affinity for chitin, a ß-1,4-linkedpolymer of N-acetylglucosamine. An intact binding domain, CBP20-N,showed a higher affinity than a C-terminal truncated domain,Ac-AMP2. The formation of a pyroglutamate residue from N-terminalGln of CBP20-N increased the affinity. The single replacementof any aromatic residue of Ac-AMP2 with Ala resulted in a significantreduction in affinity, suggesting the importance of the completeset of three aromatic residues in the ligand binding site. Themutations of Phe18 of Ac-AMP2 to the residues with larger aromaticrings, i.e. Trp, ß-(1-naphthyl)alanine or ß-(2-naphthyl)alanine,enhanced the affinity, whereas the mutation of Tyr20 to Trpreduced the affinity. The affinity of an HD for chitin mightbe improved by adjusting the size and substituent group of stackingaromatic rings.     

The structural consequences of exchanging tryptophan and tyrosine residues in B.stearothermophilus lactate dehydrogenase

A mutant Bacillus stearothermophilus lactate dehydrogenase hasbeen prepared in which all three tryptophan residues in thewild-type enzyme have been replaced by tyrosines. In addition,a tyrosine residue has been mutated to a tryptophan, which actsas a fluorescence probe to monitor protein folding. The mutantenzyme crystallizes in the same crystal form as the wild-type.The crystal structure of the mutant has been determined at 2.8Å resolution. Solution studies have suggested that thereis little effect upon the mutant enzyme as judged by its kineticproperties. Comparison of the crystal structures of the mutantand wild-type enzymes confirms this conclusion, and revealsthat alterations in structure in the region of these mutationsare of a similar magnitude to those observed throughout thestructure, and are not significant when compared with the errorsin atomic positions expected for a structure at this resolution.     

Possible role of two phenylalanine residues in the active site of human cytidine deaminase

Site-directed mutagenesis on human cytidine deaminase (CDA)was employed to mutate specifically two highly conserved phenylalanineresidues, F36 and F137, to tryptophan; at the same time, theunique tryptophan residue present in the sequence at position113 was mutated to phenylalanine. These double mutations wereperformed in order to have for each protein a single tryptophansignal for fluorescence studies relative to position 36 or 137.The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F,showed by circular dicroism and thermal stability an overallstructure not greatly affected by the mutations. The titrationof Trp residues by N-bromosuccinimide (NBS) suggested that residueW113 of the wild-type CDA and W36 of mutant F36W/W113F are buriedin the tertiary structure of the enzyme, whereas the residueW137 of mutant F137W/W113F is located near the surface of themolecule. Kinetic experiments and equilibrium experiments withFZEB showed that the residue W113 seems not to be part of theactive site of the enzyme whereas the Phe/Trp substitution inF36W/W113F and F137W/W113F mutant enzymes had a negative effecton substrate binding and catalysis, suggesting that F137 andF36 of the wild-type CDA are involved in a stabilizing interactionbetween ligand and enzyme.     

Hydrogen storage in trimethylamine hydrate: Thermodynamic stability and hydrogen storage capacity of hydrogen+trimethylamine mixed semi-clathrate hydrate

The thermodynamic stability and hydrogen occupancy for the hydrogen+trimethylamine mixed semi-clathrate hydrate system were investigated by means of phase equilibrium (pressure–temperature) measurements and Raman spectroscopic analyses. The hydrogen molecule gradually advanced to occupy the empty small cage of trimethylamine hydrate in proportion to pressure increase. Almost all small cages were filled up with the hydrogen molecules at about 80 MPa. Isothermal Raman spectroscopic analysis showed that the absorption-rate of hydrogen to the pre-treated trimethylamine hydrate was comparable to those of tetrahydrofuran hydrate. Only one hydrogen molecule was enclosed with one small cage at the equilibrium state in pre-treated trimethylamine hydrate.     

Elucidation of the role of Ser329 and the C-terminal region in the catalytic activity of Pseudomonas stutzeri L-rhamnose isomerase

Pseudomonas stutzeri l-rhamnose isomerase (l-RhI) is capable of catalyzing the isomerization between various aldoses and ketoses, showing high catalytic activity with broad substrate-specificity compared with Escherichia coli l-RhI. In a previous study, the crystal structure of P. stutzeri l-RhI revealed an active site comparable with that of E. coli l-RhI and d-xylose isomerases (d-XIs) with structurally conserved amino acids, but also with a different residue seemingly responsible for the specificity of P. stutzeri l-RhI, though the residue itself does not interact with the bound substrate. This residue, Ser329, corresponds to Phe336 in E. coli l-RhI and Lys294 in Actinoplanes missouriensis d-XI. To elucidate the role of Ser329 in P. stutzeri l-RhI, we constructed mutants, S329F (E. coli l-RhI type), S329K (A. missouriensis d-XI type), S329L and S329A. Analyses of the catalytic activity and crystal structure of the mutants revealed a hydroxyl group of Ser329 to be crucial for catalytic activity via interaction with a water molecule. In addition, in complexes with substrate, the mutants S329F and S329L exhibited significant electron density in the C-terminal region not observed in the wild-type P. stutzeri l-RhI. The C-terminal region of P. stutzeri l-RhI has flexibility and shows a flip-flop movement at the inter-molecular surface of the dimeric form.     

The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein

The 144 amino acid gene 5 protein of bacteriophage Pf1 bindstightly and cooperatively to single-stranded DNA during replicationof the phage genome. It has been suggested that aromatic aminoacid side chains are important for this interaction, probablythrough base stacking with the DNA. We have analysed the accessibilityof tyrosine residues in the DNA—protein complex, and theirimportance to the DNA-binding activity of the protein, by chemicalmodification and protection experiments using tetranitromethane.Tyrosines 21, 30 and 55 are surface accessible in the free proteinbut are protected from modification in the complex with phageDNA. Moreover, modification of these residues in the free proteinabolishes the ability to bind to DNA or oligonucleotides, asjudged by fluorescence spectroscopy and gel retardation analysis.Modification of the protein also results in the formation ofan intersubunit covalent cross-link between Tyr55 and Phe76,suggesting that Phe76 is located within the DNA-binding cleftof the protein. It is proposed that residues 17–34 ofthe Pf1 gene 5 protein form a beta-hairpin analogous to the‘DNA-binding wing’ of the fd and Ike gene 5 proteins.We suggest the existence of a single-stranded DNA binding motif,in which Tyr30 of the Pf1 protein is equivalent to the functionallyimportant Tyr26 of the fd gene 5 protein.     

Modulating functional loop movements: the role of highly conserved residues in the correlated loop motions

Loop flexibility in enzymes plays a vital role in correctly positioning catalytically important residues. This strong relationship between enzyme flexibility and function provides an opportunity to engineer new substrates and inhibitors. It further allows the design of site-directed mutagenesis experiments to explore enzymatic activity through the control of flexibility of a functional loop. Earlier, we described a novel mechanism in which a small loop triggers the motions of a functional loop in three enzymes (beta-1,4-galactosyltransferase, lipase, and enolase) unrelated in sequence, structure, or function. Here, we further address the question of how the interactions between various flexible loops modulate the movements of the functional loop. We examine beta-1,4-galactosyltransferase as a model system in which a Long loop undergoes a large conformational change (moves in space up to 20 A) upon substrate binding in addition to a small loop (Trp loop) that shows a considerably smaller conformational change. Our molecular-dynamics simulations carried out in implicit and explicit solvent show that, in addition to these two loops, two other neighboring loops are also highly flexible. These loops are in contact with either the Long loop or the Trp loop. Analysis of the covariance of the spatial displacement of the residues reveals that coupled motions occur only in one of these two loops. Sequence analysis indicates that loops correlated in their motions also have highly conserved residues involved in the loop-loop interactions. Further, analysis of crystal structures and simulations in explicit water open the possibility that the Trp loop that triggers the movement of the Long loop in the unbound conformation may also play the same role in the substrate-bound conformation through its contact with the conserved and correlated third loop. Our proposition is supported by the observation that four of the five conserved positions in the third loop are at the interface with the Trp loop. Evolution appears to select residues that drive the functional Long loop to a large conformational change. These observations suggest that altering selected loop-loop interactions might modulate the movements of the functional loop.     

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphatedehydrogenase, Ser¹⁴⁸ – Ala, was produced byoligonucleotide-directedmutagenesis. The study of the catalytic properties of this mutanthas shown that this mutation significantly affects the Michaelisconstant of inorganic phosphate and to a lesser extent thatof 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate.This result is consistent with model-building studies whichshow that, for the phosphorylation step of catalysis, inorganicphosphate must bind to the anion recognition site designatedP_i with the C(3) phosphate of the acyl-enzyme intermediate inthe alternative anion site P_s. Studies of the enantiomeric specificityusing D- and L-glyceraldehyde as substrates show that the hydroxylgroup of Ser¹⁴⁸, combined with the presence of the C(3) phosphateof the substrate, enhances stereospecificity as well as catalysis.However, the stereospecific effect cannot be a consequence ofthe direct interaction of Ser¹⁴⁸ with the C(2)-hydroxyl of thesubstrate. The changed K_m for glyceraldehyde-3-phosphate suggeststhat the initial step of hemithioacetal formation may take placewith its C(3) phosphate bound in the P_i site. This supportsthe molecular mechanism proposed by Moody (1984). Therefore,catalysis could be enhanced through interactions of the serinehydroxyl group not only with inorganic phosphate but also withthe C(3) phosphate of glyceraldehyde-3-phosphate.     

稳定我国钙镁磷肥生产的几点建议

1加强宣传稳定钙镁磷肥生产的重大意义因高浓度肥料不断发展,不少地方不重视钙镁磷肥,加上原料资金等有关问题,不少厂纷纷下马,这是极其危险的信号,必须给以应有的重视。1)认识钙镁磷肥是个好肥料。如湖南省泸溪少数民族一山田,多年施用尿素,667 m2产稻谷由400 kg降至200 kg以下;改施钙镁磷肥100 kg后,当年稻谷产量恢复至200 kg以上。这是因钙镁磷肥使板结的土壤恢复了团粒结构之故,即使高浓度肥料长期施用,也会破坏土壤团粒结构,土壤板结很难避免。这是施肥水平很高的日本,至今仍生产和进口我国钙镁磷肥的重要原因。2)钙镁磷肥属弱碱性,可…