Check out our New Publishers' Select for Free Articles

BBA - Proteins and Proteomics (v.1784, #4)

Editorial Board (pp. ii).

Stability against temperature of Sulfolobus solfataricus elongation factor 1α, a multi-domain protein by Vincenzo Granata; Giuseppe Graziano; Alessia Ruggiero; Gennaro Raimo; Mariorosario Masullo; Paolo Arcari; Luigi Vitagliano; Adriana Zagari (pp. 573-581).

The elongation factors (EF-Tu/EF-1α) are universal proteins, involved in protein biosynthesis. A detailed characterization of the stability against temperature of SsEF-1α, a three-domain protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus is presented. Thermal denaturation of both the GDP-bound ( SsEF-1α•GDP) and the ligand-free (nf SsEF-1α) forms was investigated by means of circular dichroism and fluorescence measurements, over the 4.0–7.5 pH interval. Data indicate that the unfolding process is cooperative with no intermediate species and that the few inter-domain contacts identified in the crystal structure of SsEF-1α play a role also at high temperatures . Finally, it is shown that the enzyme exhibits two different interchangeable thermally denatured states, depending on pH.

Keywords: Abbreviations; EF; elongation factor; Sc; Saccharomyces cerevisiae; Ss; Sulfolobus solfataricus; nf; nucleotide-free; CD; circular dichroism; DSC; differential scanning calorimetry; ANS; 8-anilino-1-naphthalenesulfonic acidElongation factor; Archaea; Protein stability; Thermal denaturation; Circular dichroism; Fluorescence

Modeling and experimental analyses reveal a two-domain structure and amino acids important for the activity of aminoglycoside resistance methyltransferase Sgm by Gordana Maravić Vlahoviček; Sonja Čubrilo; Karolina L. Tkaczuk; Janusz M. Bujnicki (pp. 582-590).

Methyltransferases that carry out posttranscriptional N⁷-methylation of G1405 in 16S rRNA confer bacterial resistance to aminoglycoside antibiotics, including kanamycin and gentamicin. Genes encoding enzymes from this family (hereafter referred to as Arm, for aminoglycoside resistance methyltransferases) have been recently found to spread by horizontal gene transfer between various human pathogens. The knowledge of the Arm protein structure would lay the groundwork for the development of potential resistance inhibitors, which could be used to restore the potential of aminoglycosides to act against the resistant pathogens. We analyzed the sequence–function relationships of Sgm MTase, a member of the Arm family, by limited proteolysis and site-directed and random mutagenesis. We also modeled the structure of Sgm using bioinformatics techniques and used the model to provide a structural context for experimental results. We found that Sgm comprises two domains and we characterized a number of functionally compromised point mutants with substitutions of invariant or conserved residues. Our study provides a low-resolution (residue-level) model of sequence–structure–function relationships in the Arm family of enzymes and reveals the cofactor-binding and substrate-binding sites. These functional regions will be prime targets for further experimental and theoretical studies aimed at defining the reaction mechanism of m⁷G1405 methylation, increasing the resolution of the model and developing Arm-specific inhibitors.

Keywords: Antibiotic resistance; Methylation; Modeling; Site-directed mutagenesis

Crystal structure of a myotoxic Asp49-phospholipase A₂ with low catalytic activity: Insights into Ca²⁺-independent catalytic mechanism by Luiz C. Corrêa; Daniela P. Marchi-Salvador; Adélia C.O. Cintra; Suely V. Sampaio; Andreimar M. Soares; Marcos R.M. Fontes (pp. 591-599).

A myotoxic Asp49-phospholipase A₂ (Asp49-PLA₂) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA₂ PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA₂s. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA₂ from B. jararacussu) and other Asp49-PLA₂s. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA₂s, making a hydrogen bond with the atom Oδ2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na⁺ ions at the Ca²⁺-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA₂s which are able to bind Ca²⁺ ions are also able to bind Na⁺ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA₂s indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA₂s.

Keywords: Phospholipase A; ₂; Bothrops jararacussu; venom; Myotoxic; Ca; ²⁺; -independent enzymatic activity; X-ray crystallography

Effects of pH on protein–protein interactions and implications for protein phase behavior by André C. Dumetz; Aaron M. Chockla; Eric W. Kaler; Abraham M. Lenhoff (pp. 600-610).

The effects of pH on protein interactions and protein phase behavior were investigated by measuring the reduced second osmotic virial coefficient ( b₂) for ovalbumin and catalase, and the aggregate and crystal solubilities for ovalbumin, β-lactoglobulin A and B, ribonuclease A and lysozyme. The b₂ trends observed for ovalbumin and catalase show that protein interactions become increasingly attractive with decreasing pH. This trend is in good agreement with ovalbumin phase behavior, which was observed to evolve progressively with decreasing pH, leading to formation of amorphous aggregates instead of gel bead-like aggregates, and spherulites instead of needle-like crystals. For both acidic and basic proteins, the aggregate solubility during protein salting-out decreased with decreasing pH, and contrary to what is commonly believed, neither aggregate nor crystal solubility had a minimum at the isoelectric point. β-Lactoglobulin B was the only protein investigated to show salting-in behavior, and crystals were obtained at low salt concentrations in the vicinity of its isoelectric point. The physical origin of the different trends observed during protein salting-in and salting-out is discussed, and the implications for protein crystallization are emphasized.

Keywords: Protein aggregation; Gelation; Protein crystallization; Osmotic second virial coefficient; Hydration effects

Proteome analysis of X-ray irradiated human erythroleukemia cells by Enrica Eleuterio; Fabrizio Di Giuseppe; Marilisa Sulpizio; Viviana di Giacomo; Monica Rapino; Amelia Cataldi; Carmine Di Ilio; Stefania Angelucci (pp. 611-620).

In order to discover molecular biomarkers in radiation response we investigated the effects of X-radiation on radioresistant K562 cells by using a comparative proteomic analysis. In treated cells 29 up-regulated and 10 down-regulated proteins were detected by image analysis and identified by mass spectrometry. Elongation factor 1 alpha 1 and stress-70 protein showed a 6.2 and 5.4 fold increase respectively in treated cells. Additional proteins such us pi and omega classes glutathione transferases, ATP synthase D chain, were also found to be up-regulated, suggesting that the enzyme belonging to the cellular detoxification system against oxidative stress and energetic metabolism may have a key role in the cellular response to radiation injury. This data set may provide a useful tool to design a combined chemo- and radiotherapic strategy against leukemia disease.

Keywords: Erythroleukemia cell; X-radiation; Radioresistance; Stress-70 protein; Glutathione transferase; Elongation factor 1 alpha 1; Carbohydrate metabolism

Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum by Narumi Aoki; Akie Sakiyama; Kimiko Kuroki; Katsumi Maenaka; Daisuke Kohda; Masanobu Deshimaru; Shigeyuki Terada (pp. 621-628).

Habu ( Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca²⁺ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.

Keywords: Abbreviations; CRISP; cysteine-rich secretory protein; MALDI-TOF-MS; matrix-assisted laser desorption ionization time-of-flight mass spectrometry; P; ₆₀; 60%; ammonium sulfate fraction; PSP94; prostatic secretory protein of 94 amino acids; PSPBP; PSP94-binding protein; SCP; sperm coating glycoprotein; SSP; small serum proteinCRISP; PSP94; SSP; Snake serum; Triflin; Trimeresurus flavoviridis

Effect of mutation of lysine-120, located at the entry to the active site of O-acetylserine sulfhydrylase-A from Salmonella typhimurium by Chia-Hui Tai; Wael M. Rabeh; Rong Guan; Klaus D. Schnackerz; Paul F. Cook (pp. 629-637).

O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis ofl-cysteine, the replacement of the β-acetoxy group of O-acetyl-l-serine (OAS) by a thiol. The enzyme undergoes a conformational change to close the site upon formation of the external Schiff base (ESB) with OAS. Mutation of K120 to Q was predicted to destabilize the closed form of the ESB and decrease the rate. The K120Q mutant enzyme was prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and³¹P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. Spectra suggest a shift in the tautomeric equilibrium toward the neutral enolimine and an increase in the rate of interconversion of the open and closed forms of the enzyme. A decrease in the rate of both half reactions likely reflects the stabilization of the ESB as a result of the increased rate of equilibration of the open and closed forms of the enzyme along the reaction pathway. Data suggest a role of K120 in helping to stabilize the closed conformation by participating in a new hydrogen bond to the backbone carbonyl of A231.

Keywords: O-acetylserine sulfhydrylase; Pyridoxal 5-phosphate; Spectroscopy; P-31 NMR; Site-directed mutagenesisAbbreviations; AA; α-aminoacrylate intermediate; ISB; internal Schiff base: ESB, external Schiff base; PLP; pyridoxal 5′-phosphate; HEPES; N; -(2-hydroxyethyl)piperazine-; N; '-2-ethanesulfonic acid; Mes; 2-(; N; -morpholino)ethanesulfonic acid; Ches; 2-(; N; -cyclohexylamino)ethanesulfonic acid; Mops; 3-(; N; -morpholino)propanesulfonic acid; Taps; 3-[[tris(hydroxymethyl)]amino]propanesulfonic acid; OAS; O; -acetyl-; l; -serine; TNB; 5-thio-2-nitrobenzoate; DTNB; 5,5′-dithiobis-(2-nitrobenzoate); SDS-PAGE; sodium dodecyl sulfate poyacrylamide gel electrophoresis; EDTA; ethylenediaminetetraacetate.

Oligomerization of ribonuclease A under reducing conditions by Giovanni Gotte; Massimo Libonati (pp. 638-650).

By lyophilization from 40% acetic acid solutions, bovine ribonuclease A forms well characterized, three-dimensional domain-swapped oligomers: dimers, trimers, tetramers, and higher order multimers. Each oligomeric species consists of at least two conformers. Identical oligomers also form by thermally-inducing the oligomerization of highly concentrated RNase A dissolved in fluids endowed with various denaturing power. Now, our question is: which might the influence of a reducing agent be on RNase A oligomerization, i.e., of conditions that decrease the stability of the protein and increase the mobility of its swapping domains? To address this question, we carried out experiments of RNase A oligomerization in the presence of increasing concentrations of dithiothreitol (DTT) under the two experimental conditions mentioned above. Results indicate that RNase A oligomers similar to those previously known form anyhow, but with a change of their relative proportions. The amounts of dimers and trimers decrease by increasing the concentration of DTT, while the yields of two tetramers remarkably increase. Moreover, in the presence of DTT RNase A forms labile and probably unstructured aggregates that can possibly drive the protein towards precipitation when the reducing agent's concentration increases. Taken together, these results point out once again (i) the important role of the 3D domain swapping mechanism in protein oligomerization, and (ii) the importance of the native structure of RNase A (and of proteins in general) in preventing an uncontrolled aggregation and precipitation in a reducing and highly crowded environment like that existing in a living cell.

Keywords: Abbreviations; RNase A; ribonuclease A; N-dimer and C-dimer; N-terminal and C-terminal swapped dimers, respectively; NC-trimer and C-trimer; NC-terminal (linear) or C-terminal (cyclic) swapped trimers, respectively; N; _D; N-dimer; C; _D; C-dimer; NC; _T; NC-trimer; C; _T; C-trimer; TT; tetramer(s); ddH; ₂; O; twice-distilled water, EtOH, ethanol; TFE; 2,2,2-trifluoroethanol; GdnHCl; guanidine hydrochloride; GSH; reduced glutathione; DTT; dithiothreitolRibonuclease A; RNase A oligomer; 3D domain-swapping; Reducing environment; DTT effects on RNase A oligomerization

Deswapping bovine odorant binding protein by Roberto Ramoni; Silvia Spinelli; Stefano Grolli; Virna Conti; Elisa Merli; Christian Cambillau; Mariella Tegoni (pp. 651-657).

The X-ray structure of bovine Odorant Binding Protein (bOBP) revealed its association as a domain swapped dimer. bOBP, devoid of any cysteines, contrasts with other mammalian OBPs, which are monomeric and possess at least one disulfide bridge. We have produced a mutant of bOBP in which a glycine residue was inserted after position 121. This mutation yielded a monomeric bOBP-121Gly+ in which domain swapping has been reverted. Here, we have subsequently introduced two mutations, Trp64Cys and His155Cys, in view to stabilize the putative monomer with a disulfide bridge. We have determined the crystal structure of this triple mutant at 1.65 Å resolution. The mutant protein is monomeric, stabilized by a disulfide bridge between Trp64Cys and His155Cys, with a backbone superimposable to that of native bOBP, with the exception of the hinge and of the 10 residues at the C-terminus. bOBP triple mutant binds 1-amino-anthracene, 1-octen-3-ol (bOBP co-purified ligand) and other ligands with μM Kd values comparable to those of the swapped dimer.

Keywords: Abbreviations; AMA; 1-amino-anthracene; OBP; odorant binding protein; OBP; porcine OBP; bOBP; bovine OBP; rmsd; root mean square difference; PBP; pheromone binding protein; ESRF; European Synchrotron Radiation Facility; IPTG; isopropyl β-; d; -thiogalactosideCrystal structure; Olfaction; Odorant binding protein; Domain swapping

Right-handed 14-Helix in β³-Peptides fromL-Aspartic Acid Monomers by Kamaljit Kaur; Tara Sprules; Wael Soliman; Reem Beleid; Sahar Ahmed (pp. 658-665).

β-Peptides made fromL-aspartic acid monomers form a new class of β³-peptides. Here we report the first three-dimensional NMR solution structure of a β³-hexapeptide (1) fromL-aspartic acid monomers in 2,2,2-trifluoroethanol (TFE). We show that1 forms a right-handed 14-helical structure in TFE. α-peptides from naturally occurringL-amino acids adopt a right-handed α-helix whereas β³-peptides formed from β³-amino acids derived from naturally occurringL-amino acids form left-handed 14-helices. The right-handed 14-helical conformation of1 is a better mimic of α-peptide conformations. Using the NMR structure of1 in TFE, we further study the conformation of1 in water, as well as two similar β³-peptides (2 and3) in water and TFE by molecular dynamics (MD) simulations. NMR and MD results suggest loss of secondary structure of1 in water and show that it forms a fully extended structure.2 and3 contain residues with oppositely charged side chains that engage in salt-bridge interactions and dramatically stabilize the 14-helical conformation in aqueous media.

Keywords: Abbreviations; CD; circular dichroism; MD; molecular dynamics; NMR; nuclear magnetic resonance spectroscopy; NOE; nuclear Overhauser effect; NOESY; two-dimensional NOE spectroscopy; ns; nanosecond; pbc; periodic boundary conditions; ps; picosecond; RMSD; root mean square deviation; TFE; 2,2,2-trifluoroethanol; SPC; simple point chargeβ-Peptides; 14-Helical conformation; Solution structure; MD simulations

Binding of polycyclic aromatic hydrocarbons to mutants of odorant-binding protein: A first step towards biosensors for environmental monitoring by Yin Wei; Anna Brandazza; Paolo Pelosi (pp. 666-671).

Polycyclic aromatic hydrocarbons are among the most threatening pollutants widely present in the environment. Simple and economic methods of continuous monitoring of these compounds in real time are not yet available, although becoming increasingly needed. Odorant-binding proteins (OBPs) present unique characteristics of thermal and chemical stability for building robust, reliable, and inexpensive biosensors for such molecules. To investigate this possibility, we have engineered the pig OBP, whose three-dimensional structure has been resolved, introducing a tryptophan residue in the core of the binding pocket, as a fluorescence reporter for the presence of bound ligands. Binding affinities of several polyaromatic hydrocarbons to mutagenically modified OBPs were measured in competitive binding assays. Moreover, the presence of aromatic ligands was also successfully monitored in the modified OBPs by recording the quenching of intrinsic fluorescence of the protein. These data indicate that OBPs bind several aromatic polycyclic compounds with good affinities, that the specificity of these proteins can be easily modified by changing specific amino acid residues and that the introduction of a tryptophan residue in the binding site allows monitoring of aromatic ligands using direct fluorescence measurements.

Keywords: Abbreviations; OBP; odorant-binding protein; 1-AMA; 1-aminoanthracene; PAH; polycyclic aromatic hydrocarbonOdorant-binding proteins; Polyaromatic hydrocarbons; Intrinsic fluorescence; Pollutants; Biosensors

Sub-domains of the dystrophin rod domain display contrasting lipid-binding and stability properties by Sébastien Legardinier; Jean-François Hubert; Olivier Le Bihan; Christophe Tascon; Chantal Rocher; Céline Raguénès-Nicol; Arnaud Bondon; Serge Hardy; Elisabeth Le Rumeur (pp. 672-682).

Dystrophin is a muscle scaffolding protein that establishes a structural link between the cytoskeleton and the extracellular matrix. Despite the large body of knowledge about the dystrophin gene and its interactions, the functional importance of the large central rod domain remains highly controversial. It is composed of 24 spectrin-like repeats interrupted by four hinges that delineate three sub-domains. We express repeat 1–3 and repeat 20–24 sub-domains, delineated by hinges 1–2 and 3–4 and the single repeats 2 and 23. We determine their lipid-binding properties, thermal and urea stabilities and refolding velocities. By using intrinsic tryptophan fluorescence spectroscopy and size exclusion chromatography, we show that repeat 2 and the repeat 1–3 sub-domain strongly interact with anionic lipids. By contrast, repeat 23 and the repeat 20–24 sub-domain do not interact with lipids. In addition, the repeat 1–3 sub-domain and repeat 2 are dramatically less stable and refold faster than the repeat 20–24 sub-domain and repeat 23. The contrasting properties of the two sub-domains clearly indicate that they make up two units of the rod domain that are not structurally interchangeable, thus providing molecular evidence supporting the observations on the biological function of dystrophin.

Keywords: Abbreviations; DOPC; dioleyl-phosphatidylcholine; DOPS; dioleyl-phosphatidylserine; DOPE; dioleyl-phosphatidylethanolamine; LB; Luria-Bertani; MLV; multilamellar vesicles; PAGE; polyacrylamide gel electrophoresis; SDS; Sodium dodecyl sulphate; SUVs; small unilamellar vesiclesDystrophin rod domain; Dystrophin repeat; Lipid binding; Thermal unfolding; Urea unfolding

Copper-induced structural changes in the ovine prion protein are influenced by a polymorphism at codon 112 by Sujeong Yang; Alana M. Thackray; Tim J. Fitzmaurice; Raymond Bujdoso (pp. 683-692).

Prion diseases are associated with conformational change in the copper-binding protein PrP. The copper-binding sites in PrP are located in the N-terminal region of the molecule and comprise a series of tandem repeats of the sequence PHGGGWGQ together with two histidines at residues 96 and 111 (human PrP numbering). The co-ordination of copper ions within the non-octapeptide repeat metal ion-binding site involves Met109 (human numbering, which corresponds with Met112 in ovine PrP) and the binding of copper to this site leads to an increase in β-sheet formation in PrP. Here we have investigated the influence of the M112T polymorphism on copper-induced structural changes in ovine recombinant PrP. M112ARQ and T112ARQ ovine PrP show similar secondary structure although M112ARQ appears more thermostable than T112ARQ. Following treatment with copper, M112ARQ showed a greater increase in β-sheet content than did T112ARQ when measured by CD spectroscopy and by ELISA using anti-PrP monoclonal antibodies. These biochemical and biophysical differences between M112ARQ and T112ARQ correlate with similar differences seen between allelic variants of ovine PrP associated with susceptibility and resistance to classical scrapie. These observations suggest that T112ARQ may provide a measure of resistance to classical scrapie pathogenesis compared to M112ARQ.

Keywords: PrPC; Polymorphism; Transmissible spongiform encephalopathies; β-sheet; Conformational stability

Comparative insight into the Zn(II)-, Cd(II)- and Cu(I)-binding features of the protozoan Tetrahymena pyriformis MT1 metallothionein by Jordi Domènech; Roger Bofill; Anna Tinti; Armida Torreggiani; Sílvia Atrian; Mercè Capdevila (pp. 693-704).

Tetrahymena pyriformis MT1 (TpyMT1) is a model among ciliate metallothioneins (MTs). Here, we report on the analytic (ICP-AES, GC-FPD), spectroscopic (CD, UV–Vis, Raman) and spectrometric (ESI-MS) characterization of its recombinant Cd(II)-, Zn(II)- and Cu(I)-complexes, and of those formed during in vitro Zn/Cd and Zn/Cu replacement. In the presence of Cd(II), TpyMT1 renders a major Cd₁₁–TpyMT1 species, which is also the final step reached in the in vitro Zn/Cd exchange process in Zn₁₁–TpyMT1. Spectroscopic data supports a different folding of the isostoichiometric Cd₁₁– and Zn₁₁–TpyMT1 complexes. Unexpectedly, TpyMT1 biosynthesis in Zn(II)-rich cultures was sensitive to the aeration degree, so that high oxygenation rendered undermetalated, partially-oxidized, complexes (Zn₉–TpyMT1). Biosynthesis in Cu(I)-rich media rendered extremely heterogeneous mixtures of Cu_xZn_y-species ( x+ y=8–20), where the higher the aeration, the higher the Zn(II) content. The complexity of these samples was reproduced during the Zn/Cu replacement, as the number of generated species increased gradually with the addition of copper to Zn₁₁–TpyMT1. According to our results, a clear preference of TpyMT1 for Cd(II) binding, rather than for Zn(II), and especially Cu(I) can be postulated. This character is totally consistent with the induction pattern of the TpyMT1 gene and the postulated role of TpyMT1 in Cd-detoxification.

Keywords: Abbreviation; TpyMT1; T. pyriformis; MT1 Tetrahymena; Metallothionein; Metal-binding; Raman spectroscopy; Sulfide ions

An analytical method for determining relative specificities for sequential reactions catalyzed by the same enzyme: General formulation by David Alexander Mitchell; Frédéric Carrière; Nadia Krieger (pp. 705-715).

We present a general formulation of a model that can be used to analyze reaction profiles in systems in which a single enzyme catalyzes several sequential reactions with the same molecular backbone. The analysis of these so-called “repeated-attack systems” allows estimation of the specificities that the enzyme has for the various intermediate substrates that appear in the reaction mixture, relative to the specificity that it has for the initial substrate. Our analytical method has the important advantage that it is not affected by competitive or uncompetitive inhibition, nor by denaturation of the enzyme during the reaction. We carry out case studies in three different systems, the lipase-catalyzed alcoholysis of triacylglycerols, the phytase-catalyzed removal of phosphate groups from phytic acid and the β-amylase-catalyzed removal of maltose units from maltoheptaose. Our model fits well to all reaction profiles in which the phenomenon of processivity does not occur. It can therefore be used as a general tool for characterizing the relative specificities of “repeated-attack enzymes”.

Keywords: Specificity constant; Repeated-attack; Sequential hydrolysis; Enzyme kinetics; Mathematical model; Hydrolase

Two bacterial collagenolytic serine proteases have different topological specificities by Yoshiko Uesugi; Jiro Arima; Hirokazu Usuki; Masaki Iwabuchi; Tadashi Hatanaka (pp. 716-726).

From among 2000 soil isolates, we purified a secreted serine protease from Streptomyces omiyaensis (SOT), which has extremely high gelatinolytic activity. Using sequence analysis, the primary structure of SOT showed 77% identity with that of S. griseus trypsin (SGT). We constructed recombinants SOT and SGT using S. lividans. They indicated similar properties on optimum pH and temperature, thermostability, and substrate preference using fluorescence energy transfer combinatorial libraries. SOT greatly hydrolyzed both type I and type IV collagens, but SGT has poor ability to hydrolyze type IV collagen. Furthermore, SOT exhibits higher hydrolytic activities toward other protein substrate such as gelatin and casein than SGT. These results suggest that these two enzymes have different topological specificities in spite of their similar primary structures. We also constructed chimeras between SOT and SGT to investigate which domain is associated with differences in their substrate specificity. In comparison to substrate specificities of chimeras, we found that the N-terminal domain contributes to the determination of topological specificity.

Keywords: Abbreviations; SOT; Streptomyces omiyaensis; serine protease; SGT; Streptomyces griseus; trypsin; FRETS; fluorescence energy transfer substrateTrypsin; Collagenolytic enzyme; Serine protease; Streptomyces; FRETS

Sections

Personal tools

BBA - Proteins and Proteomics (v.1784, #4)

Sections

Personal tools

Local resources

BBA - Proteins and Proteomics (v.1784, #4)