共查询到20条相似文献,搜索用时 15 毫秒
1.
Aita T 《Journal of theoretical biology》2008,254(2):252-263
We obtained several structural features of an NK fitness landscape by analytical approach. Particularly, we focused on spatial distributions of “ascending slopes”, “highlands”, “nearly neutral networks”, and “local optima” along the fitness coordinate W, from the viewpoint of adaptive walks with step-width d , where d is the number of mutated sites (Hamming distance) after a generation. The parameter k governs the degree of the ruggedness on the NK landscape, and we handled cases where k is moderate against the sequence length. From the foot up to the middle region on the landscape, many ascending slopes exist (high evolvability) and these slopes extend up near the “highland”, which is mathematically defined as the specific region where the expectation of the fitness increment becomes zero. Denoting the standard deviation of the fitness change at by SD*, we considered the existence of “nearly neutral networks”, which percolate in the fitness band between W-SD* and W+SD*. Our results suggest that the highland corresponds to a phase-transition threshold of the formation of the nearly neutral networks. Near or over the highland, “local optima at the dth order” appear drastically (low evolvability), where d means the radius of their basins. The value of increases with d increasing. Then, as the fitness (=altitude) becomes higher, the basin size of the local optima increases. This leads to a conclusion that it is very hard or impossible for walkers with step-width d to reach near the global peak when d is a realistic large value: d=1-6, and suggests that the region over the middle in real landscapes may be considerably smooth with small k-values to maintain high evolvability. 相似文献
2.
Maristela Pereira Zhihong Song Ludier Kesser Santos-Silva Mathew H. Richards Thi Thuy Minh Nguyen JiaLin Liu Celia Maria de Almeida Soares Aline Helena da Silva Cruz Kulothungan Ganapathy W. David Nes 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2010,1801(10):1163-1174
The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502 Da. The enzymatic C24-methylation gave a Kmapp of 38 μM and kcatapp of 0.14 min−1. Quite unexpectedly, “plant” cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-13C]- or [24-2H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of 1H and 13CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Δ24(25)-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (Ki 14 nM) and 26,27-dehydrolanosterol (Ki 54 μM and kinact of 0.24 min−1) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC50, 30 nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C1-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT. 相似文献
3.
Sandra Eklund Ann-Christin Lindås Emil Hamnevik Mikael Widersten 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(4):561-570
Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of kcatapp/KM probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the KM and kcatapp are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of kcatapp, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases. 相似文献
4.
Keeble AH Joachimiak LA Maté MJ Meenan N Kirkpatrick N Baker D Kleanthous C 《Journal of molecular biology》2008,379(4):745-759
Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with Kd values that vary between 10− 4 and 10− 14 M, characteristics that are explained by a ‘dual-recognition’ mechanism. In this work, we addressed for the first time the energetics of Im protein recognition by colicin DNases through a combination of E9 DNase alanine scanning and double-mutant cycles (DMCs) coupled with kinetic and calorimetric analyses of cognate Im9 and non-cognate Im2 binding, as well as computational analysis of alanine scanning and DMC data. We show that differential ΔΔGs observed for four E9 DNase residues cumulatively distinguish cognate Im9 association from non-cognate Im2 association. E9 DNase Phe86 is the primary specificity hotspot residue in the centre of the interface, which is coordinated by conserved and variable hotspot residues of the cognate Im protein. Experimental DMC analysis reveals that only modest coupling energies to Im9 residues are observed, in agreement with calculated DMCs using the program ROSETTA and consistent with the largely hydrophobic nature of E9 DNase-Im9 specificity contacts. Computed values for the 12 E9 DNase alanine mutants showed reasonable agreement with experimental ΔΔG data, particularly for interactions not mediated by interfacial water molecules. ΔΔG predictions for residues that contact buried water molecules calculated using solvated rotamer models met with mixed success; however, we were able to predict with a high degree of accuracy the location and energetic contribution of one such contact. Our study highlights how colicin DNases are able to utilise both conserved and variable amino acids to distinguish cognate from non-cognate Im proteins, with the energetic contributions of the conserved residues modulated by neighbouring specificity sites. 相似文献
5.
Aita T Hayashi Y Toyota H Husimi Y Urabe I Yomo T 《Journal of theoretical biology》2007,246(3):538-550
We have developed a methodology for extracting characteristic properties of a fitness landscape of interest by analyzing fitness data on an in vitro molecular evolution. The in vitro evolution is required to be conducted as the following "adaptive walk": a single parent sequence generates N mutant sequences as its offsprings, and the fittest individual among the N offsprings will become a new parent in the next generation. N is the library size of mutants to be screened in a single generation. Our theory of the adaptive walk on the "NK landscape" suggests the following: the adaptive walker starting from a random sequence climbs the landscape easily in an early stage, and then reaches a stationary phase in which the mutation-selection-random drift balance sets in. The stationary fitness value is nearly proportional to square root of ln N. Our analysis is performed from the following points: (1) stationary fitness values, (2) time series of fitness in the transitional state, (3) mutant's fitness distribution, and (4) the strength of selection pressure. Applying our methodology, we analyzed experimental data on the in vitro evolution of a random polypeptide (139 amino acids) toward acquiring infectivity (= ability to infect) of fd phage. As a result, we estimated that k is about 27 in this system, indicating that an arbitrary residue in a sequence is affected from other 23% residues. In this article, we demonstrated that the experimental data is consistent with our theoretical equations quantitatively, and that our methodology for extracting characteristic properties of a fitness landscape may be effective. 相似文献
6.
A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn2+ that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a kcat of 15,500 min−1 and a Km of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 Å of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased kcat of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in Km, suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in kcat, but had little effect on Km. 相似文献
7.
Morillas M Eberl H Allain FH Glockshuber R Kuennemann E 《Journal of molecular biology》2008,376(3):721-735
The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and kcat/KM values up to 5 × 105 s−1 M−1. The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pKa1 = 6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k2 = 4.4±0.6 s−1 and k3 = 1.6±0.5 s−1, respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities. 相似文献
8.
Many organic anions bind free Ca2+, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (Kapp) describing the Ca2+ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca2+ electrodes coupled to either a 3 M KCl or a Na+ selective electrode were used to redetermine Kapp. All solutions contained 130 mM Na+, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl− that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of Kapp if left uncorrected. Because the Na+ concentration in all solutions was constant, a Na+ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of Kapp either after correction for these LJP changes or via direct reference to a Na+ electrode showed that SCFAs do not bind Ca2+ and that the Kapp for the binding of Ca2+ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM. 相似文献
9.
In adaptation biology the discovery of intracellular osmolyte molecules that in some cases reach molar levels, raises questions of how they influence protein thermodynamics. We've addressed such questions using the premise that from atomic coordinates, the transfer free energy of a native protein (ΔGtr, N) can be predicted by summing measured water-to-osmolyte transfer free energies of the protein's solvent exposed side chain and backbone component parts. ΔGtr, D is predicted using a self avoiding random coil model for the protein, and ΔGtr, D − ΔGtr, N, predicts the m-value, a quantity that measures the osmolyte effect on the N ? D transition. Using literature and newly measured m-values we show 1:1 correspondence between predicted and measured m-values covering a range of 12 kcal/mol/M in protein stability for 46 proteins and 9 different osmolytes. Osmolytes present a range of side chain and backbone effects on N and D solubility and protein stability key to their biological roles. 相似文献
10.
The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2–11 and at temperature 90 °C for 2 1/2 h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE .The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425 Da. The secondary structure of API as analyzed by the CD spectra showed 7% α-helix, 49% β-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin–API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC50 and Ki values 4.0 nM and (3.83 nM–5.31 nM) respectively. The overall inhibition constant Ki? value is 0.107 ± 0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I ? (k4, k5) EI ? (k6, k7) EI?. Rate constant k6 = 2.73 ± 0.32 s− 1 reveals a fast isomerization of enzyme–inhibitor complex and very slow dissociation as proved by k7 = 0.068 ± 0.009 s− 1. The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI?. 相似文献
11.
Kinetic characterizations of protein translocation on DNA are nontrivial because the simultaneous presence of multiple different mechanisms makes it difficult to extract the information specific to a particular translocation mechanism. In this study, we have developed new approaches for the kinetic investigations of proteins' sliding and intersegment transfer (also known as “direct transfer”) in the target DNA search process. Based on the analytical expression of the mean search time for the discrete-state stochastic model, we derived analytical forms of the apparent rate constant kapp for protein-target association in systems involving competitor DNA and the intersegment transfer mechanism. Our analytical forms of kapp facilitate the experimental determination of the kinetic rate constants for intersegment transfer and sliding in the target association process. Using stopped-flow fluorescence data for the target association kinetics along with the analytical forms of kapp, we have studied the translocation of the Egr-1 zinc-finger protein in the target DNA association process. Sliding was analyzed using the DNA-length-dependent kapp data. Using the dependence of kapp on the concentration of competitor DNA, we determined the second-order rate constant for intersegment transfer. Our results indicate that a major pathway in the target association process for the Egr-1 zinc-finger protein is the one involving intersegment transfer to a nonspecific site and the subsequent sliding to the target. 相似文献
12.
Yuki Gomibuchi Taro Q.P. Uyeda Takeyuki Wakabayashi 《Biochemical and biophysical research communications》2013
Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å2 to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (Vmax) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller Kapp) than that of the wild-type actin, with the Vmax being almost unchanged. The Kapp and Vmax of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of Kapp was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/Kapp reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release. 相似文献
13.
The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (kcat/Km) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0–9.0 and 50 °C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 °C and 60 °C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 °C to 75 °C and caused a prominent 31-fold increase in kcat/Km with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency. 相似文献
14.
Demian Riccardi Shuo Yang Qiang Cui 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(2):342-351
Recent QM/MM analyses of proton transfer function of human carbonic anhydrase II (CAII) are briefly reviewed. The topics include a preliminary analysis of nuclear quadrupole coupling constant calculations for the zinc ion and more detailed analyses of microscopic pKa of the zinc-bound water and free energy profile for the proton transfer. From a methodological perspective, our results emphasize that performing sufficient sampling is essential to the calculation of all these quantities, which reflects the well solvated nature of CAII active site. From a mechanistic perspective, our analyses highlight the importance of electrostatics in shaping the energetics and kinetics of proton transfer in CAII for its function. We argue that once the pKa for the zinc-bound water is modulated to be in the proper range (~ 7.0), proton transfer through a relatively well solvated cavity towards/from the protein surface (His64) does not require any major acceleration. Therefore, although structural details like the length of the water wire between the donor and acceptor groups still may make a non-negligible contribution, our computational results and the framework of analysis suggest that the significance of such “fine-tuning” is likely secondary to the modulation of pKa of the zinc-bound water. We encourage further experimental analysis with mutation of (charged) residues not in the immediate neighborhood of the zinc ion to quantitatively test this electrostatics based framework; in particular, Φ analysis based on these mutations may shed further light into the relative importance of the classical Grotthus mechanism and the “proton hole” pathway that we have proposed recently for CAII. 相似文献
15.
María Carmen Pallarés Carlos Marcuello Laura Botello-Morte Andrés González María Francisca Fillat Anabel Lostao 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(3):623-631
Fur (ferric uptake regulator) proteins are involved in the control of a variety of processes in most prokaryotes. Although it is assumed that this regulator binds its DNA targets as a dimer, the way in which this interaction occurs remains unknown. We have focused on FurA from the cyanobacterium Anabaena sp. PCC 7120. To assess the molecular mechanism by which FurA specifically binds to “iron boxes” in PfurA, we examined the topology arrangement of FurA–DNA complexes by atomic force microscopy. Interestingly, FurA–PfurA complexes exhibit several populations, in which one is the predominant and depends clearly on the regulator/promoter ratio on the environment. Those results together with EMSA and other techniques suggest that FurA binds PfurA using a sequential mechanism: (i) a monomer specifically binds to an “iron box” and bends PfurA; (ii) two situations may occur, that a second FurA monomer covers the free “iron box" or that joins to the previously used forming a dimer which would maintain the DNA kinked; (iii) trimerization in which the DNA is unbent; and (iv) finally undergoes a tetramerization; the next coming molecules cover the DNA strands unspecifically. In summary, the bending appears when an “iron box” is bound to one or two molecules and decreases when both “iron boxes” are covered. These results suggest that DNA bending contributes at the first steps of FurA repression promoting the recruitment of new molecules resulting in a fine regulation in the Fur-dependent cluster associated genes. 相似文献
16.
Kato K Takeuchi H Ohki T Waki M Usami K Hassan H Clausen H Irimura T 《Biochemical and biophysical research communications》2008,371(4):698-701
Interaction of Vicia villosa agglutinin-B4 (VVA-B4) to glycopeptides with O-linked GalNAc residues was investigated by surface plasmon resonance. The affinity was shown to be influenced by the arrangement of O-glycosylation sites on a peptide, PTTTPITTTTK, representing the tandem repeat of MUC2. The association rate constant was relatively high with a particular category of GalNAc-peptides in which more than three amino acid residues were placed between GalNAc-Thr residues. PTT∗T∗PITT∗T∗TK (T∗ indicates GalNAc-Thr) had the highest association rate constant among the glycopeptides tested. The dissociation rate constant was low in the peptides containing consecutive GalNAc residues and PT∗TTPIT∗T∗T∗TK was the lowest of the glycopeptides tested. Dissociation constant (KD), calculated as kd/ka was the lowest with PTT∗T∗PITT∗T∗TK. Therefore, the arrangement but not the quantity of GalNAc residues apparently determines the affinity between VVA-B4 and peptides with attached GalNAc residues. 相似文献
17.
Emily M.S. Hopwood Duale AhmedSusan M. Aitken 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(2):465-472
Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4–1.2 pH units, likely due to repositioning of the cofactor and modification of the pKa of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3–3, 26–58 and 124–568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The Kml-Cth of E333 substitution variants is increased ~ 17-fold, while Kml-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (kcat/Kml-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (kcat/Kml-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (kcat/Kml-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity. 相似文献
18.
Matthew J. Grisewood Nathanael P. Gifford Robert J. Pantazes Ye Li Patrick C. Cirino Michael J. Janik Costas D. Maranas 《PloS one》2013,8(10)
OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., kcat or kcat/KM) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that KM correlates (R2 = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, kcat/KM correlates (R2 = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and kcat correlates (R2 = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved KM, kcat, and kcat/KM for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving KM, kcat, or kcat/KM. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S. 相似文献
19.
20.
In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-β-alanine to β-alanine, is catalyzed by two β-alanine synthase (βASase, EC 3.5.1.6) subfamilies. We show that the “prototype” eukaryote βASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-βA compared with a representative of fungal βASases, the yeast Saccharomyces kluyveri βASase, which has a high Km value (71 mM). S. kluyveri βASase is specifically inhibited by dipeptides and tripeptides, and the apparent Ki value of glycyl-glycine is in the same range as the substrate Km. We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal βASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors. 相似文献