The purification and properties of an aryl beta-hexosidase from bovine liver.

An aryl β-hexosidase was purified 800-fold from bovine liver. The purified enzyme hydrolyzed p-nitrophenyl glycosylpyranoside derivatives of β-d-galactose, β-d-glucose, β-d-xylose, β-d-mannose, and α-l-arabinose, but did not hydrolyze several other p-nitrophenyl glycosides. The enzyme also catalyzed hydrolysis of a variety of plant arylglucosides. Disaccharides, polysaccharides, glycolipids, glycoproteins, and glycosaminoglycans containing terminal nonreducing β-d-galactopyranosyl or β-d-glucopyranosyl residues were not hydrolyzed. The pH optima for the several substrates tested ranged from 7.0 to 9.5. The purified enzyme was homogeneous by disc gel electrophoresis and had a molecular weight of 41,000 by Sephadex gel filtration and 46,000 by disc gel electrophoresis performed in the presence of sodium dodecyl sulfate. The enzyme readily transferred glycosyl residues from susceptible β-galactosides or β-glucosides to other sugars; the resulting products were not hydrolyzed by the enzyme. Methyl α-d-glucopyranoside was the most efficient carbohydrate acceptor compound tested. The enzyme exhibited a K_m for p-nitrophenyl β-d-galactopyranoside of 1.78 × 10^?3m and for p-nitrophenyl β-d-glucopyranoside, 2.50 × 10^?3m when incubations were conducted in the presence of 0.15 m methyl α-d-glucopyranoside. Aryl β-hexosidase was found in the cytosol of all mammalian livers tested, but could not be detected in liver of birds, reptiles, or fish; low levels were detected in frog liver. Analysis of bovine extracts indicated that the enzyme occurred in liver, kidney, and intestinal mucosa; it was not detected in testis, spleen, serum, or muscle.     

Enzymatic synthesis of p-nitrophenyl 35-O-β-N-acetylglucosaminyl|-α-maltopentaoside by lysozyme; a novel substrate for human amylase assay

Transglycosylation from di-N-acetylchitobiose to the 3-position at the nonreducing end glucosyl group of p-nitrophenyl α-maltopentaoside was regioselectively induced through the use of hen egg-white lysozome. The enzyme formed p-nitrophenyl 3⁵-O-β-N-acetylglucosaminyl-α-maltopentaoside (5% of the enzyme-catalyzed net decreased of p-nitrophenyl α-maltopentaoside) from di-N-acetylchitobiose as a donor and p-nitrophenyl α-maltopentaoside as an acceptor. The rate of the transglycosylation depended on the concentration of substrate, the temperature and the pH. The hydrolytic actions of human pancreatic and salivary α-amylase on this derivative were examined. The maltopentaoside derivative was shown to be useful as a substrate for α-amylase assay through a coupled reaction involving α-D-glucosidase and glucoamylase.     

Isolation of β-N-acetyl-d-hexosaminidases from lupin seed

A rapid procedure for the isolation of β-N-acetyl-d -hexosaminidase from lupin seed meal is described. This involves affinity chromatography of a seed extract on concanavalin A, followed by chromatography on DEAE-Sepharose. The purified enzyme was obtained in three forms, hexosaminidases A, B and B₁, capable of hydrolysing both p-nitrophenyl β-2-acetamido-2-deoxy-d-glucopyranoside and p-nitrophenyl β-2-acetamido-2-deoxy-d-galactopyranoside. Enzyme A was relatively less active towards the galactosaminide substrate, than were the B forms of the enzyme.     

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

Introduction of non-natural amino acid residues into the substrate-specific P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors

A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P₁ position was synthesized by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted in para position of the phenyl ring, l-pyridylalanine and N-4-nitrobenzylgycine. Their inhibitory activities with bovine α-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants (K_a). All analogues inhibited bovine α-chymotrypsin. The highest inihbitory activity displayed peptides with the fluorine, nitro and methyl substituents. They were 13–15-fold more active than [Phe⁵]SFTI-1 used as a reference. They are the most potent chymotrypsin inhibitors of this size. Substitution of Lys5 by Phe did not change the cathepsin G inhibitory activity. Introduction of Phe(p-F), Phe(p-NH₂) and Phe(p-CH₃) in this position retained the affinity towards this proteinase, whereas Phe(p-guanidine) gave an inhibitor more than twice as active, which appeared to be stable in human serum. On the other hand, a peptomeric analogue with N-4-nitrobenzylglycine failed to inhibit cathepsin G. Despite the fact the introduced amino acids were non-coded, the peptide bonds formed by them were hydrolyzed by chymotrypsin. We postulate that additional interaction of para-substitutents with the enzyme are responsible for the enhanced inhibitory activity of the analogues.     

Purification and characterization of novel cutinase from nasturtium (Tropaeolum majus) pollen.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.     

An examination of the rate assay to determine the active site normality of papain

Data are presented which demonstrate that the α-N-benzoyl-l-argine ethyl ester rate assay procedure, based on a burst titration with N-benzyloxy-carbonyl-l-tyrosine p-nitrophenyl ester as previously desribed (1), is an accurate and reliable method for determining the normality of papain in solution.     

A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides

A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p-nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p-nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal β-N-acetylhexosaminidase using p-nitrophenyl-β-2-acetamido-2-deoxy-d-glucopyranoside (NPADG) at pH 5.0. A number of different wavelengths may be used for the assay depending upon the criterion of the user; maximum sensitivity at a selected pH, determination of enzyme pH optima with a pH-independent difference extinction coefficient, or the reduction of background absorbance for kinetic studies at high substrate concentrations.     

An improved method for determining the activity of benzoyl-arginine p-nitroanilide-hydrolyzing enzyme in crude tissue extracts

The method described in this communication is sensitive and allows the direct determination in crude tissue extracts of the enzyme that hydrolyzes α-N-benzoyl-dl-arginine p-nitroanilde (Bz-Arg-NPhNO₂). It may be useful in determining cathepsin B activity in crude enzyme preparations when the reaction mixture is incubated with an inhibitor of trypsinlike enzymes which are also capable of hydrolyzing Bz-Arg-NPhNO₂.     

The first α-1,3-glucosidase from bacterial origin belonging to glycoside hydrolase family 31

Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES–GroEL chaperones. Recombinant LJAG31 was an α-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of k_cat/K_m) toward nigerose, maltulose, and kojibiose than other natural substrates having an α-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial α-1,3-glucosidase to be characterized with a high k_cat/K_m value for nigerose [α-d-Glcp-(1 → 3)-d-Glcp]. Transglucosylation of 4-nitrophenyl α-d-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl α-nigeroside and 4-nitrophenyl α-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) α-1,3-glucosidase of glycoside hydrolase family 31.     

Effect of hydroxynitrobenzylation of tryptophan-177 on reactivity of active site cysteine-25 in papain

It is known that the enzymatic activity of papain (EC 3.4.22.2) toward α-N-benzoyl-l-arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4-nitrophenyl (N-carbobenzoxy)glycinate (S.-M. T. Chang and H. R. Horton, 1979, Biochemistry18, 1559–1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactivation by S_N2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloroacetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulfhydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the increased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, α-N-benzoyl-l-phenylalanyl-l-valyl-l-arginine p-nitroanilide, and toward an ester substrate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxynitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward α-N-benzoyl-l-arginine p-nitroanilide, apparently by blocking nonproductive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159).     

An apparatus for safe and convenient handling of anhydrous, liquid hydrogen fluoride at controlled temperatures and reaction times. Application to the generation of oligosaccharides from polysaccharides

β-d-Gal-(1 → 4)-β-d-GlcNAc-OC₆H₄NO₂-p (p-nitrophenyl N-acetyl-β-lactosaminide) and β-d-Gal-(1 → 6)-β-d-GlcNAc-OC₆H₄NO₂-p (p-nitrophenyl N-acetyl-β-isolactosaminide) were regioselectively synthesized from lactose and p-nitrophenyl 2-acetamido-2-deoxy-glucopyranoside, employing transglycosylation by the β-d-galactosidase from Bacillus circulans and by controlling the concentration of organic solvent in the reaction system. The (1 → 4)-linked disaccharide was formed exclusively when the concentration of organic solvent was high, whereas the (1 → 6)-linked isomer was produced with a low concentration. Further utilization of the transglycosylation by the enzyme led to the regioselective formation of β-d-Gal-(1 → 4)-d-GalNAc and β-d-Gal-(1 → 4)-β-d-GalNAc-OC₆H₄NO₂-p. With the enzyme, β-d-galactosyl transfer occurred preferentially at the O-4 position of GlcNAc and GalNAc, regardless of the configuration of the hydroxyl group.     

Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves : A Steady-State and Pre-Steady-State Study

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k_cat and k_cat/K_m reflect the ionization of two groups with pK_a values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K_m are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10⁻¹⁰ molar at the optimum pH value (approximately 7.5).     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

α-Galactosidase from Alfalfa Seeds (Medicago sativa L.)

An α-galactosidase from alfalfa seeds was purified 140-fold by ammonium sulfate fractionation, and column chromatography on Sephadex G-100, DEAE- and CM-Sephadex. Polyacrylamide-gel electrophoresis of the purified enzyme showed a single protein band. The molecular weight was estimated to be approximately 57,000 by gel-filtration. The purified enzyme hydrolyzed p-nitrophenyl α-d-galactoside more rapidly than raffinose. The maximal enzyme activities were obtained at pH 4.0 and 5.5 for p-nitrophenyl α-d-galactoside and at 4.5 for raffinose. The enzyme was shown to be inhibited by Hg²⁺ and Ag⁺ ions, and d-galactose.     

A sensitive fluorometric assay for plasminogen, plasmin, and streptokinase

A rapid, convenient, and highly sensitive fluorometric assay for plasmin (P), plasminogen (Pg), and streptokinase (SK) as the activator complex (SK·P) is described. These assays are based on the measurement of the fluorescence of β-naphthol (βN) released from α-N-methyl α-N-tosyl-l-lysine β-naphthol ester (MTLNE) by the P present or generated during the assay. The rate of βN release may be followed by direct recording or determined subsequently, following termination of the enzyme reaction at a fixed digestion time, by the addition of p-nitrophenyl-p′-guanidinobenzoate. The latter method may be readily automated. The K_m and V values for the hydrolysis of MTLNE by P or SK·P are equivalent. The Pg activator activity of P was shown to be very small (less than 0.2% of that of SK·P).     

Importance of secondary enzyme-substrate interactions in human cathepsin G and chymotrypsin II catalysis

The active center of human leukocyte cathepsin G, human pancreatic chymotrypsin II, and bovine α-chymotrypsin has been investigated with a series of substrates of general formula succinyl-(l-alanine)_n-phenylalanine-p-nitroanilide (n = 0 to 3). The three proteinases have an extended substrate binding site which includes at least six subsites. Secondary interactions are very important for their catalytic power since the longest substrate is hydrolyzed 600 to 1100 times faster than the shortest one. The regulatory subsite is S₄ for bovine α-chymotrypsin and human cathepsin G whereas it is S₅ for human chymotrypsin II. Cathepsin G is a poor catalyst compared to the two other enzymes.     

Kinetics and mechanism of catalysis by proteolytic enzymes: The kinetics of hydrolysis of derivatives of l-lysine and S-(beta-aminoethyl)-l-cysteine(thialysine)by bovine trypsin

1. Several esters of the α-N-toluene-p-sulphonyl and N-benzoyl derivatives of l-lysine and S-(β-aminoethyl)-l-cysteine have been synthesized. 2. The kinetics of hydrolysis of the esters by bovine trypsin have been compared. Values of k₀ are similar for corresponding derivatives of the isosteric amino acids and deacylation of an acyl-enzyme appears to be rate-determining in each case. There are, however, some quantitative kinetic differences between the various series of substrates.     

The assay of the bromelains using N alpha-CBZ-L-lysine p-nitrophenyl ester and N-CBZ-glycine p-nitrophenyl ester as substrates

Two new esterolytic assays of the pineapple stem bromelains are described. They use as substrates the p-nitrophenyl esters of N^α-CBZ-l-lysine (CLN) and N-CBZ-glycine (CGN). The activity is monitored by the direct spectrophotometric measurement of the enzyme-catalyzed hydrolysis of these esters at 340 nm. The bromelains are rapidly activated with 1 mm l-cysteine at pH 4.6 for the CLN assay and pH 6.1 for the CGN assay. EDTA has no measurable effect. The sensitivities of the assays approach 10 μg/ml in a reaction time of 3 min.     

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.