Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)₂) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)_n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)_4–6 and produced (GlcNAc)_2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)₅ to produce (GlcNAc)₂ (79.2%) and (GlcNAc)₃ (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)_n (n = 2–4) was pNp-(GlcNAc)₂ >> pNp-(GlcNAc)₄ > pNp-(GlcNAc)₃. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)_n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.     

Metal-ion-catalyzed hydrolysis of monomethyl and dimethyl esters of 3-(2-imidazoleazo)phthalic acid: Bifunctional catalysis by carboxyl group and the metal ion in the hydrolysis of an alkyl ester

The Cu(II) or Ni(II) ion-catalyzed hydrolysis of methyl 2-carboxy-6-(2-imidazoleazo)benzoate (1) and the corresponding dimethyl ester (2) was studied kinetically at various pH values. For 2, the ester group located at the o position to the azo substiuent was hydrolyzed. From the rate data obtained at various metal concentrations, the values of k_cat and K_f were estimated at each pH value. For the Ni(II)-catalyzed hydrolysis of 1 at pH < 4, k_cat increases as pH is lowered, indicating bifunctional catalysis by the carboxyl group and the metal ion. For most of the reactions investigated under other conditions, the ester hydrolysis was subjected to sole catalysis by the metal ions. Detailed analysis of kinetic data obtained for these reactions indicated that the metal-ion catalysis involves the rate-determining breakdown of the tetrahedral intermediates formed by the addition of a water molecule or hydroxide ion. The bifunctional catalysis by the carboxyl group and Ni(II) ion can be considered as a model for carboxypeptidase A. The kinetic data indicate that the bifunctional catalysis proceeds through the nucleophilic attack of the carboxylate ion at the Ni(II)-coordinated carbonyl group.     

Study of Secondary Specificity of Enteropeptidase in Comparison with Trypsin

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu) _n -Lys(Arg)--B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G₅DK-F(NO₂)G (k _cat/K _m = 2380 mM^–1·min^–1) is more efficient than the fusion protein PrAD₄K-P26 (k _cat/K _m = 1260 mM^–1·min^–1).     

A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)₆. Although the binding of tri-N-acetylchitotriose, (GlcNAc)₃, to OEL perturbed several backbone resonances in the ¹H–¹⁵N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pK_a values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)₃ binding. Thermal unfolding experiments and modeling study of (GlcNAc)₆ hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites ?3 and ?2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.     

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.)

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23000–28000 and isoelectric points 10.3–10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-d-glucosamine oligosaccharide (GlcNAc)₃. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8–100% acetylated chitosans and (GlcNAc)_4–6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)_2–6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (Ab_PLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.Mention of a trademark, warranty, propriety, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations Ab antibody - BCLVC basic chitinase/lysozyme cv. Valencia callus - BCVC basic chitinase cv. Valencia callus - CE capillary electrophoresis - CM-chitin-RBV carboxymethyl-chitin-remazol brilliant violet - GlcNAc N-acetyl-d-glucosamine - HEWL hen egg-white lysozyme - M_r relativemolecular mass - pI isoelectric point - PLC potato leaf chitinase - PR pathogenesis-related - SEC size exclusion chromatography We thank Mr. M. Burkhart, Ms. T.-T. Ho, and Ms. M. Doherty for their valuable technical assistance. A portion of the funding for this work was made available from the Citrus Production Research Marketing Order by the Division of Marketing and Development, Florida Department of Agriculture and Consumer Services, Bob Crawford, Commissioner.     

Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF) (AT). Model peptideswith truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic parameters ofenteropeptidase hydrolysis for these substrates were determined.K _m values for all substrates with truncated linker (10^-3 M) are an order of magnitude higher thancorresponding values for typical enteropeptidase artificial peptide or fusion protein substrates with full enteropeptidase linker –DDDDK– (K _m 10^-4 M). k _cat values for AT, Hb (2–8), WDDRG and WDDKG are 30–40 min^-1. But one additional amino acid residue at both N- and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency: k _cat value for Hb (1–9) is 1510 min^-1. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active peptides in vitro along with its unique natural substrate trypsinogen was demonstrated.     

OptZyme: Computational Enzyme Redesign Using Transition State Analogues

OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., k_cat or k_cat/K_M) 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 K_M correlates (R² = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, k_cat/K_M correlates (R² = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and k_cat 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 K_M, k_cat, and k_cat/K_M for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving K_M, k_cat, or k_cat/K_M. 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.     

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6¹,6³- and 6¹,6⁴-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the k_cat and K_m values for transglycosylation were 1.78 × 10³ s⁻¹ and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10³ s⁻¹ and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the k_cat/K_m ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.     

N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

We used quench flow to study how N⁶-methylated adenosines (m⁶A) affect the accuracy ratio between k_cat/K_m (i.e. association rate constant (k_a) times probability (P_p) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated k_cat/K_m for Glu-tRNA^Glu, EF-Tu and GTP forming ternary complex (T₃) reading cognate (GAA and Gm⁶AA) or near-cognate (GAU and Gm⁶AU) codons. k_a decreased 10-fold by m⁶A introduction in cognate and near-cognate cases alike, while P_p for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated k_cat/K_m for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um⁶AA) and near-cognate (UAG and Um⁶AG) stop codons to decrease 6-fold or 3-fold by m⁶A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m⁶A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate k_cat/K_m, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate k_cat/K_m.     

Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation

This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9₂₅-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9₂₅-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5′-product terminating in a 2′,3′-phosphodiester and a 3′-product terminating in a 5′-OH. Under single turnover conditions at 24°C, 9₂₅-11t displays a maximum first-order rate constant, k_cat, of 0.037 min⁻¹ and a catalytic efficiency, k_cat/K_m, of 5.3 × 10⁵ M⁻¹ min⁻¹. The measured value of k_cat under catalyst excess conditions agrees with the value of k_cat observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9₂₅-11t was gauged in terms of k_cat values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k_cat values by a factor of 4 to 250, indicating that 9₂₅-11t displays significant sequence specificity despite the lack of an apparent Watson–Crick base-pairing scheme for recognition.     

Ficin-Catalyzed Reactions. Hydrolysis of alpha-N-Benzoyl-l-Arginine Ethyl Ester and alpha-N-Benzoyl-l-Argininamide

The effect of pH on the hydrolysis of α-N-benzoyl-l-arginine ethyl ester (BAEE) and α-N-benzoyl-l-argininamide (BAA) by a proteolytic enzyme component purified from Ficus carica var. Kadota latex has been studied in detail over the pH range of 3 to 9.5. k_cat (lim) values for the hydrolysis of BAEE and BAA were essentially identical (5.20 and 5.01 sec⁻¹, respectively at 30°). k_cat values for hydrolysis of BAEE and BAA were dependent on prototropic groups with apparent pK values of 4.24 and 8.53 and 4.10 and 8.59, respectively. k_cat (lim) values for tht hydrolysis of BAEE and BAA were essentially identical (5.20 and groups of pK 4.33 and 8.60 and 4.55 and 8.51, respectively. Thus the pH optimum is 6.5 for both substrates. K_m (app) values for BAEE and BAA were 3.32 × 10⁻²m and 6.03 × 10⁻²m respectively over the pH range of 3.9 to 8.0. These data are interpreted in terms of the involvement of a carboxyl and a sulfhydryl group in the active center of the enzyme. The data do not support the concept that deacylation of the acyl-enzyme is completely the rate controlling step in the hydrolyses. Rather, it appears that the magnitude of k₂ and k₃ are not greatly different.     

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k_cat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s^–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k_cat for ATP hydrolysis by 7-fold, to 0.32 s^–1, while not significantly altering the apparent K_m. The specificity constant for the interaction between ATP and topo IIβ (k_cat/K_mapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.     

Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn²⁺. Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K_m of 32.2 μM and k_cat of 29.5 s⁻¹ with Ala-pNA and a K_m of 11.1 μM and k_cat of 9.49 s⁻¹ with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.     

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galβ1→3GlcNAc, LNB) and galacto-N-biose (Galβ1→3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBP_Bl). In the presence of α-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k _cat/K _m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBP_Bl showed similar k _cat/K _m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-α-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.     

Polyclonal Antibodies from Hen Egg Yolk (IgY) with Hydrolysis Activity

Polyclonal catalytic antibodies (abzymes) play an important role in immunology research. In this study, we report polyclonal antibodies IgYs isolated from chicken egg yolk with hydrolysis activity for the first time. The IgYs were raised in hens using HNPBV [4-(hydroxy (naphthalen-2-yloxy) phosphoryl) butanoic acid] attached to BSA (Bovine serum albumin) as an immunogen. Anti-(HNPBV-BSA) IgYs were isolated from yolks of the eggs laid using a two-step salt precipitation and one-step gel filtration protocol. NA (naphthalen-2-yl acetate) was selected as the substrate and the hydrolysis reaction of the IgYs for it was examined. The result reveals that the rate of the hydrolysis reaction is higher (K _cat/K _uncat∼2 × 10⁴). The purified IgYs were digested with pepsin and the smaller fragment (Fab′) with specific antigen binding properties was produced. The research indicates that the enzymatic properties of Fab′ are similar to IgYs. The catalytic activity of the IgYs was further determined by measuring the rate of hydrolysis of NA in the presence of inhibitor. These findings show that chicken egg is an excellent donor for polyclonal catalytic antibodies.     

Studies on Bacillus licheniformis endo-\-1,3-1,4-d-glucanase: characterization and kinetic analysis

Summary A new purification procedure for endo-\-1,3-1,4-d-glucanase from Bacillus licheniformis is described. The secreted enzyme was purified both from B. licheniformis and from recombinant Escherichia coli harbouring the cloned gene by ion exchange chromatography on a CM-Sepharose matrix at pH 5.6. The mature enzyme was resistant to proteolysis by trypsin and chymotrypsin but it was slowly digested by protease V8. It showed a continuous trimming where no large-limit polypeptides were noticeable thus supporting a monodomain structure. Former appearing peptides have been assigned theoretically according to the protein sequence and predictive methods of accessible areas. Kinetic parameters for the hydrolysis of barley \-glucan and lichenan by measuring the net release of reducing sugars at the optimum pH (7.02) and temperature (55° C) are k _cat=3500 ±800 s^–1 (turnover number) and K _m=1.45±0.21 mg/ml for barley \-glucan and k _cat=3000±750 s^–1 and K _m=1.98±0.40 mg/ml for lichenan. Correspondence to: E. Querol     

Characterization of site‐directed mutants of residues R58, R59, D116, W340 and R372 in the active site of E. coli cystathionine β‐lyase

Cystathionine β‐lyase (CBL) catalyzes the hydrolysis of L ‐cystathionine (L ‐Cth) to produce L ‐homocysteine, pyruvate, and ammonia. A series of active‐site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k_cat/K for the β‐elimination reaction range from a reduction of only 3‐fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L ‐Cth is: R372 >> R58 > W340 ≈ R59 > D116. Comparison of the kinetic parameters for L ‐Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the α‐carboxylate moiety. The increase in the pK_a of the acidic limb and decrease in the pK_a of the basic limb of the k_cat/K versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pK_a of an active‐site residue.     

Inter‐conversion of catalytic abilities in a bifunctional carboxyl/feruloyl‐esterase from earthworm gut metagenome

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k_cat/K_m)]_CE/[(k_cat/K_m)]_FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys²⁸¹, Asp²⁸², Asn³¹⁶ and Lys³¹⁷) situated close to the catalytic core (Ser¹⁴³/Asp²⁷³/His³⁰⁵) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k_cat/K_m values) and enzymes with inverted specificity ((k_cat/K_m)_CE/(k_cat/K_m)_FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol⁻¹), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol⁻¹) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.     

STUDIES ON THE KINETICS OF ACETYLCHOLINESTERASE IN THE RESISIANT AND SUSCEPTIBLE STRAINS OF HOUSEFLY (MUSCA DOMESTICA)

Abstract Acetylcholinesterase (AChE) in the susceptible (S) and the resistant (R) strains of housefly (Musca domestica) was investigated using kinetic analysis. The V_max values of AChE for hydrolyzing acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were 4578.50 and 1716.08nmol/min/mg* protein in the R strain, and were 1884.75 and 864.72 nmol/min/mg. protein in the Sstrain, respectively. The V_max ratios of R to S enzyme were 2.43 for ATCh and 1.98 for BTCh. The K_m values of AChE for ATCh and BTCh were 0.069 and 0.034 mmol/L in the S strain, and 0.156, 0.059 mmol/L in the R strain, respectively. The K_m ratios of R to S enzyme were 2.26 for ATCh and 1.74 for BTCh. The k_i ratios of S to R enzyme for three insecticides propoxur, methomyl and paraoxon were 46.04, 4.17 and 2. 86, respectively. In addition, k_cat and k_cat/K_m for measuring turnover and catalytic efficiency of AChE were determined using eserine as titrant. The k_cat values of AChE from the R strain for both ATCh and BTCh were higher than those values from the S strain. But the values of k_cat/K_m were in contrary to the k_cat values with R enzyme compared to S enzyme. The AChE catalytic properties and sensitivity to the inhibition by three insecticides in the R and S strains of housefly were discussed based on contribution of V_max, K_m, k_i, k_cat and k_cat/K_m. All these data implied that AChE from the R strain might be qualitatively altered. We also observed an intriguing phenomenon that inhibitors could enhance the activity of AChE from the resistant strain. This “flight reaction” of the powerful enzyme might be correlated with the developing resistance of housefly to organophosphate or carbamate insecticides.