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1.
Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus 总被引:3,自引:0,他引:3
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Gianna Palmieri Carmen Bianco Giovanna Cennamo Paola Giardina Gennaro Marino Maria Monti Giovanni Sannia 《Applied microbiology》2001,67(6):2754-2759
A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the Km value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca2+ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes. 相似文献
2.
Ernesto A. Roman Santiago E. Faraj Alexandra Cousido-Siah André Mitschler Alberto Podjarny Javier Santos 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(6):1168-1180
Adaptation of life to low temperatures influences both protein stability and flexibility. Thus, proteins from psychrophilic organisms are excellent models to study relations between these properties. Here we focused on frataxin from Psychromonas ingrahamii (pFXN), an extreme psychrophilic sea ice bacterium that can grow at temperatures as low as − 12 °C. This α/β protein is highly conserved and plays a key role in iron homeostasis as an iron chaperone. In contrast to other frataxin homologs, chemical and temperature unfolding experiments showed that the thermodynamic stability of pFXN is strongly modulated by pHs: ranging from 5.5 ± 0.9 (pH 6.0) to 0.9 ± 0.3 kcal mol− 1 (pH 8.0). This protein was crystallized and its X-ray structure solved at 1.45 Å. Comparison of B-factor profiles between Escherichia coli and P. ingrahamii frataxin variants (51% of identity) suggests that, although both proteins share the same structural features, their flexibility distribution is different. Molecular dynamics simulations showed that protonation of His44 or His67 in pFXN lowers the mobility of regions encompassing residues 20–30 and the C-terminal end, probably through favorable electrostatic interactions with residues Asp27, Glu42 and Glu99. Since the C-terminal end of the protein is critical for the stabilization of the frataxin fold, the predictions presented may be reporting on the microscopic origin of the decrease in global stability produced near neutral pH in the psychrophilic variant. We propose that suboptimal electrostatic interactions may have been an evolutionary strategy for the adaptation of frataxin flexibility and function to cold environments. 相似文献
3.
The oxidation of thiocyanate by iron(V) (Fe(V)) was studied as a function of pH in alkaline solutions by a premix pulse radiolysis technique. The rates decrease with an increase in pH. The rate law for the oxidation of SCN− by Fe(V) was obtained as −d[Fe(V)]/dt = k10{[H+]2/([H+]2 + K2[H+] + K2K3)}[Fe(V)][SCN−], where k10 = 5.72 ± 0.19 × 106 M−1 s−1, pK2 = 7.2, and pK3 = 10.1. The reaction precedes via a two-electron oxidation, which converts Fe(V) to Fe(III). Thiocyanate reacts approximately 103× faster with iron(V) than does with iron(VI). 相似文献
4.
Duncan E.K. SutherlandKelly L. Summers Martin J. Stillman 《Biochemical and biophysical research communications》2012,426(4):601-607
Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn2+ or Cd2+ ions into two distinct domains: an N-terminal β-domain that binds 3 Zn2+ or Cd2+, and a C-terminal α-domain that binds 4 Zn2+ or Cd2+. Although stepwise metalation to the saturated M7-MT (where M = Zn2+ or Cd2+) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (Mn-MT, n = 0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K1 < K2 < K3 < ··· < K7), weakly cooperative (where K1 = K2 = K3 = ··· = K7), and noncooperative, (where K1 > K2 > K3 > ··· > K7). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn2+ and Cd2+ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn2+ and Cd2+ binding to rhMT 1a. 相似文献
5.
Gerard Esteban Jennifer Allan Abdelouahid Samadi Andrea Mattevi Mercedes Unzeta José Marco-Contelles Claudia Binda Rona R. Ramsay 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(6):1104-1110
Monoamine oxidases (MAO) and cholinesterases are validated targets in the design of drugs for the treatment of Alzheimer's disease. The multi-target compound N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)-N-methylprop-2-yn-1-amine (ASS234), bearing the MAO-inhibiting propargyl group attached to a donepezil moiety that inhibits cholinesterases, retained activity against human acetyl- and butyryl-cholinesterases. The inhibition of MAO A and MAO B by ASS234 was characterized and compared to other known MAO inhibitors. ASS234 was almost as effective as clorgyline (kinact/KI = 3 × 106 min− 1 M− 1) and was shown by structural studies to form the same N5 covalent adduct with the FAD cofactor. 相似文献
6.
Seiichi Taguchi Akiyoshi Ozaki Haruo Momose 《Applied and environmental microbiology》1998,64(2):492-495
A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46–51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the kcat/Km value) 100% higher than that of the wild type at 10°C when N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased Km value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60°C and circular dichroism spectra at 10°C.Biological systems have evolved over billions of years to perform very specific biological functions within the context of living organisms. From the evolution of natural proteins, we have learned that proteins are highly adaptable, constantly changing biomolecules. Accordingly, we can explore the functions of protein molecules free from the constraints of a living system by mimicking some of the processes of Darwinian evolution in the test tube. We have been attempting to use “evolutionary engineering” to improve enzyme proteins for practical purposes. Evolutionary engineering can be defined as a technological alternative to protein engineering for the creation of desired enzymes based on a Darwinian sequential program of mutagenesis and selection. To date, the pioneering works have concentrated on the application of evolution engineering to the isolation of thermostable enzymes (7, 16) and organic solvent-adapted enzymes (1).Cold adaptation of enzymes would be an attractive project covering a wide range of applications, e.g., food processing, washing, biosynthetic processes with volatile intermediates, and environmental bioremediation. Very recently, extensive attempts to isolate different types of cold-adapted enzymes from psychrophilic organisms have been made by Gerday and coworkers (2, 3). In contrast, we have initiated for the first time an artificial evolution program for the cold adaptation of subtilisin BPN′, a mesophilic and industrially useful alkaline serine protease. Fortunately, the tertiary structure of subtilisin has been well established, and the enzyme is a good model to which protein engineering can be applied for alteration of its properties. However, much of the theoretical basis for designing a cold-adapted subtilisin is still unclear. If we were able to obtain a variety of cold-adapted subtilisins, rich background data on the structure-function relationship of this enzyme would be of enormous value in helping to clarify the molecular mechanism of cold adaptation.For this purpose, we originally devised an evolution system for multistep random mutagenesis connected with screening of the evolved enzymes with an Escherichia coli host vector and also established a system for enzyme overproduction with a Bacillus subtilis host vector (14) to allow enzymatic analysis of the evolvants. In the present communication, we describe our improved evolution system and the isolation and characterization of a cold-adapted subtilisin which exhibits activity 100% higher than that of the wild-type enzyme at 10°C. 相似文献
7.
The Complete Amino Acid Substitutions at Position 131 That Are Positively Involved in Cold Adaptation of Subtilisin BPN′
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To ascertain whether position 131 of a mesophilic protease, subtilisin BPN′, is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492–495, 1998), a full set of subtilisin BPN′ mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10°C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN′ and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (kcat/Km) at 10°C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in kcat and a decrease in Km. All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10°C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60°C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering. 相似文献
8.
We report on the characterisation of a member of the acylaminoacyl peptidase family, the first isolated from bacteria. The enzyme was obtained from the psychrophilic bacterium Sporosarcina psychrophila and shows the typical features of cold adaptation (low Tm, optimal temperature of 40 °C, poor thermal stability). It was also tested for substrate specificity, effect of metals, temperature dependence and structure stability and revealed promiscuous catalytic activity on at least two chemically distinct substrates, with kcat/Km values for ester hydrolysis and acylamino acids cleavage of 1.7 × 104 s−1 M−1 and 6.2 × 103 s−1 M−1, respectively. Despite some properties cannot be explained with current models, results report on the relevance of structural and catalytic properties for the successful adaptation to cold temperatures. 相似文献
9.
Ryuichiro Suzuki Kazue Terasawa Keitarou Kimura Zui Fujimoto Mitsuru Momma Mikihiko Kobayashi Atsuo Kimura Kazumi Funane 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(7):919-924
Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the kcat/KM value of CITase-598K was approximately two times higher (32.2 s− 1 mM− 1) than that of CITase-T3040 (17.8 s− 1 mM− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes. 相似文献
10.
Cry1Ia and Cry1Aa proteins exhibited toxicities against Prays oleae with LC50 of 189 and 116 ng/cm2, respectively. The ability to process Cry1Ia11 protoxin by trypsin, chymotrypsin and P. oleae larvae proteases was studied and compared to that of Cry1Aa11. After solubilization under high alkaline condition (50 mM NaOH), Cry1Aa11 was converted into a major fragment of 65 kDa, whereas Cry1Ia11 protoxin was completely degraded by P. oleae larvae proteases and trypsin and converted into a major fragment of 70 kDa by chymotrypsin. Using less proteases of P. oleae juice, the degradation of Cry1Ia11 was attenuated. When the solubilization (in 50 mM Na2CO3 pH 10.5 buffer) and activation were combined, Cry1Ia11 was converted into a proteolytic product of 70 kDa after 3 h of incubation with trypsin, chymotrypsin and P. oleae juice. These results suggest that the in vivo solubilization of Cry1Ia11 was assured by larval proteases after a swelling of the corresponding inclusion due to the alkalinity of the larval midgut. 相似文献
11.
Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-d-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10 000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH-kcat profile of the forward reaction has a pKa of 6.9 ± 0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation. 相似文献
12.
Variable pH 13C NMR and 1H NMR spectroscopic studies of the β-cyclodextrin (β-CD) in alkaline aqueous solutions revealed that β-CD does not deprotonate at pH < 12.0. Further increase in solution pH results in the deprotonation of OH-groups adjacent to C-2 and C-3 carbon atoms of β-CD glucopyranose units, whereas the deprotonation of OH-groups adjacent to C-6 carbon atoms is expressed less markedly. The pKa values for β-CD OH-groups adjacent to C-2 and C-3 carbon atoms are rather close, pKa1,2 being 13.5 ± 0.2 (22.5 °C). 相似文献
13.
Ona Barauskas Amoreena C. CorsaRuth Wang Scott HluhanichDebi Jin Magdeleine HungHuiling Yang William E. Delaney IVBrian E. Schultz 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays.Methods
Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods.Results
GS-9256 and vedroprevir had measured Ki values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; Ki values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was > 10,000-fold against all tested off-target proteases. Association rate constants of 4 × 105 M− 1 s− 1 and 1 × 106 M− 1 s− 1, respectively, were measured, and dissociation rate constants of 4.8 × 10− 5 s− 1 and 2.6 × 10− 4 s− 1 were determined.Conclusions
GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics.General Significance
The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C. 相似文献14.
A new psychrophilic marine protease was found from a marine bacterium Flavobacterium YS-80 in the Chinese Yellow Sea. The protease is about 49 kD with an isoelectric point about 4.5. It consists of 480 amino acids and is homologous to a psychrophilic alkaline protease (PAP) from an Antarctic Pseudomonas species. The protein was purified from the natural bacterium fermented and crystallized. Its crystal structure (PDB ID 3U1R) was solved at 2.0 Å by Molecular Replacement using a model based on PAP, and was refined to a crystallographic Rwork of 0.16 and an Rfree of 0.21. The marine protease consists of a two domain structure with an N-terminal domain including residues 37–264 and a C-terminal domain including residues 265–480. Similar to PAP, the N-terminal domain is responsible for proteolysis and the C-terminal is for stability. His186, His190, His196 and Tyr226 are ligands for the Zn2+ ion in the catalytic center. The enzyme''s Tyr226 is closer to the Zn2+ ion than in PAP and it shows a stronger Zn2+―Tyr-OH bond. There are eight calcium ions in the marine protease molecule and they have significantly shorter bond distances to their ligands compared to their counterparts in all three crystal forms of PAP. On the other hand, the loops in the marine protease are more compact than in PAP. This makes the total structure stable and less flexible, resulting in higher thermo stability. These properties are consistent with the respective environments of the proteases. The structural analysis of this new marine protease provides new information for the study of psychrophilic proteases and is helpful for elucidating the structure-environment adaptation of these enzymes. 相似文献
15.
Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu+ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔGw° = 12.9 kJ mol− 1, m = 4.1 kJ mol− 1 M− 1, Cm = 3 M, and ΔCpw° = 0.93 kJ mol− 1 K− 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures. 相似文献
16.
Our previous study has suggested that mutation of the amino acid residue Asp102 has a significant effect on the fumarate-mediated activation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME). In this paper, we examine the cationic amino acid residue Arg98, which is adjacent to Asp102 and is highly conserved in most m-NAD(P)-MEs. A series of R98/D102 mutants were created to examine the possible interactions between Arg98 and Asp102 using the double-mutant cycle analysis. Kinetic analysis revealed that the catalytic efficiency of the enzyme was severely affected by mutating both Arg98 and Asp102 residues. However, the binding energy of these mutant enzymes to fumarate as determined by analysis of the KA,Fum values, show insignificant differences, indicating that the mutation of Arg98 and Asp102 did not cause a significant decrease in the binding affinity of fumarate. The overall coupling energies for R98K/D102N as determined by analysis of the kcat/Km and KA,Fum values were −2.95 and −0.32 kcal/mol, respectively. According to these results, we conclude that substitution of both Arg98 and Asp102 residues has a synergistic effect on the catalytic ability of the enzyme. 相似文献
17.
Gisbert Grossmann Kim A. Burkov Lubov A. Myund Claudia Verwey 《Inorganica chimica acta》2004,357(3):797-808
Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H6L) have been studied by dilatometric, potentiometric and 31P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M+ + HL5− ? MHL4− and M+ + L6− ? ML5−, respectively, were determined: logKNaHL=1.08(0.07), logKKHL=0.86(0.08), logKNaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pKa5app and pKa6app. Correction formulae were derived to convert actual dissociation constants pKa into apparent dissociation constants pKaapp (or vice versa). The actual dissociation constants were found: pKa5(H2L4− ? H+ + HL5−)=7.47(0.03) and pKa6(HL5− ? H+ + L6−)=14.1(0.1). The anisotropy of 31P chemical shifts of salts MnH6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts. 相似文献
18.
Justin E. Culbertson Michael D. Toney 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):240-246
PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg2 + dependent and catalyzes a similar SN2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give Kchorismate = 20 ± 4 μM, KMg2 + = 294 ± 22 μM, KL-gln = 11 ± 1 mM, and kcat = 2.2 ± 0.2 s− 1 for a random kinetic mechanism. PhzE can use NH4+ as an alternative nucleophile, while Co2 + and Mn2 + are alternative divalent metals. 相似文献
19.
Svetlana Sharoyan Alvard Antonyan Sona Mardanyan Hayk Harutyunyan Naira Movsisyan Narek Hovnanyan Karlen Hovnanyan 《Neurochemistry international》2013
The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (Km = 37.5 μM, kcat/Km = 1.7 × 103 M−1sec−1) and Aβ42 (Km = 138.4 μM, kcat/Km = 1.90 × 102 M−1sec−1). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates. 相似文献
20.
Biochemical studies to elucidate the structural basis for xyloglucan specificity among GH12 xyloglucanases are lacking. Accordingly, the substrate specificity of a GH12 xyloglucanase from Aspergillus niger (AnXEG12A) was investigated using pea xyloglucan and 12 xylogluco-oligosaccharides, and data were compared to a structural model of the enzyme. The specific activity of AnXEG12A with pea xyloglucan was 113 μmol min−1 mg−1, and apparent kcat and Km values were 49 s−1 and 0.54 mg mL−1, respectively. These values are similar to previously published results using xyloglucan from tamarind seed, and suggest that substrate fucosylation does not affect the specific activity of this enzyme. AnXEG12A preferred xylogluco-oligosaccharides containing more than six glucose units, and with xylose substitution at the −3 and +1 subsites. The specific activities of AnXEG12A on 100 μM XXXGXXXG and 100 μM XLLGXLLG were 60 ± 4 and 72 ± 9 μmol min−1 mg−1, respectively. AnXEG12A did not hydrolyze XXXXXXXG, consistent with other data that demonstrate the requirement for an unbranched glucose residue for hydrolysis by this enzyme. 相似文献