The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide by thermolysin

The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl-l-leucine amide by thermolysin has been reinvestigated. It was found that the K_m for the enzyme substrate interaction is 2.5 × 10^?3m at pH 7.2. This K_m is an order of magnitude less than what has been previously assumed to be the K_m for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10^?3m substrate and is based on the assumption that the substrate concentration is much less than the K_m. Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10^?4m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the K_m.     

A continuous spectrophotometric assay for cyclic 3′,5′-nucleotide phosphodiesterase

In the course of studies on cyclic nucleotide phosphodiesterase, the need for an assay which was both sensitive and continuous was realized. Most of the methods available for monitoring phosphodiesterase either depend on the use of accessory enzymes, and are accordingly subject to intrinsic limitations, or are not capable of continuously monitoring the enzymatic reaction. The present paper describes a new spectrophotometric assay for cyclic nucleotide phosphotidesterase which is highly reproducible, rapid, simple, and more sensitive than many of the previously published assays for this enzyme. The method is sensitive enough to detect the enzymatic conversion of 75 pmol of cAMP to 5′-AMP per minute. This assay is based on the fact that the hydrolysis of cyclic nucleotides to the corresponding 5′-phosphate ester by phosphodiesterase makes available an additional titratable proton of the 5′-phosphate group. By incorporating phenol red into the assay mixture, the rate of proton production can be continuously measured by monitoring the decrease in absorbance of the basic chromophore of phenol red at 560 nm. The primary advantages of the spectrophotometric assay described here are: (a) it provides initial velocity measurements, and thus is ideally suited to studying the kinetic properties of partially purified preparations of enzyme, and (b) it does not require the tedious and time-consuming purification of commercially available substrates which is often required when radioisotopic assays are used in detailed kinetic studies. The chief limitations are: (a) the sensitivity is not sufficient to accurately monitor the “low K_m” enzyme, and (b) the use of the assay to quantitate revoveries throughout extensive purification, where different buffer salts at different pH values are used, would require that the enzyme be dialyzed prior to assay.     

An H-ATPase Assay: Proton Pumping and ATPase Activity Determined Simultaneously in the Same Sample

A continuous spectrophotometric assay of H⁺-ATPase activity was developed by combining two well-known methods for measuring proton pumping and ATPase activity. Proton uptake into plasma membrane vesicles from Avena sativa L. (cv Rhiannon) was monitored as the absorbance decrease at 495 nm of the ΔpH probe acridine orange. Simultaneously, ATPase activity was measured by following the absorbance decrease at 340 nanometers by coupling ATP hydrolysis enzymatically to the oxidation of NADH. This H⁺-ATPase assay is convenient for determining the relative relationship between ATP hydrolysis and proton pumping.     

Spectrophotometric assay of NADase-catalyzed reactions

A spectrophotometric method for the assay of NADase-catalyzed reactions was developed. The assay consisted of monitoring the decrease in absorbance at 275 nm accompanying the enzyme-catalyzed hydrolysis of ?-NAD. A millimolar extinction coefficient of 0.89 at 275 nm was determined for the hydrolysis of the nicotinamide-ribosidic bond of ?-NAD. Under assay conditions the assay was shown to be linear up to 50% completion. A linear relationship between the rate of ?-NAD hydrolyzed and the amount of NADase added was observed. The K_m and V_max values for Bungarus fasciatus venom NADase-catalyzed hydrolysis of ?-NAD were determined spectrophotometrically and were shown to be the same as those determined by other analytical methods.     

Assay for enzyme activity by following the absorbance change of pH-indicators

Based on the absorbance change of indicators with the concentration of hydrogen ion released from an enzyme-catalyzed reaction, a convenient colorimetric method was established for the assay of acidic phospholipase A₂ and glycogen phosphorylase b. Brilliant yellow and bromothymol blue were chosen as indicators for assays of acidic phospholipase A₂ and glycogen phosphorylase b by following the absorbance changes at 495 and 615 nm, respectively. The method is simple, sample-saving, sensitive and valid for a wide range of enzyme concentrations. It can be extended for assaying other enzymes catalyzing reactions with hydrogen ion concentration changes.     

Histidine as an essential residue in the active site of the copper enzyme galactose oxidase.

The pH dependence of the oxidation of β-methyl-d-galactopyranoside by galactose oxidase at 1.33 mm O₂ has been determined. The k_cat exhibits a bell-shaped dependence on the ionization of at least two groups in the enzyme-substrate complex, pK_b' = 6.3 and pK_a' = 7.1, respectively. The pH-independent value for k_cat at 1.33 mm O₂ (nonsaturating) and saturating glycoside is 1435 s^?; the pH optimum is 6.7. Galactose oxidase is inactivated rapidly by iodoacetamide. Although the reaction is much slower, iodoacetate also inactivates the enzyme. The inactivation by iodoacetamide obeys saturation kinetics; at pH 7.0 k₃ = 2.19 min^?1 and K_i = 5.1 mM; k₃ but not K_i exhibits a bell-shaped pH dependence, with pK_a values of 6.3 and 7.6, respectively. Labeling with [¹⁴C]iodoacetamide establishes that one carboxamidomethyl group is incorporated per enzyme molecule. This incorporation parallels the loss of enzymatic activity. Only N-3-carboxymethylhistidine is detected in chromatograms following hydrolysis of the labeled protein. The protein-bound copper is not lost as a consequence of alkylation. Apogalactose oxidase does not react with iodoacetamide. The alkylation is inhibited by the oxidation of an active center tryptophan residue (s) by N-bromosuccinimide. The fraction of residual enzyme activity remaining after tryptophan oxidation corresponds to the extent of labeling by [¹⁴C]iodoacetamide. Although alkylation causes little change in the spin Hamiltonian parameters of the Cu(II) atom, it nearly abolishes both the optical activity and optical absorbance of the metal. The native tryptophan fluorescence of the enzyme, which is a sensitive probe of its active site, is also markedly affected. Since binding of a substrate, β-methyl-d-galactopyranoside, reduces fluorescence as it does in the active enzyme and binding of CN^? at the Cu(II) site as detected by electron spin resonance appears unaffected by the alkylation, the effect of alkylation is on catalysis, per se. Both a catalytic and a subtle conformational role for the active site histidine are inferred from the results.     

Causes and elimination of erratic blanks in enzymatic metabolite assays involving the use of NAD+ in alkaline hydrazine buffers: improved conditions for the assay of L-glutamate, L-lactate, and other metabolites.

In the alkaline hydrazine buffers frequently used for enzymatic metabolite assays, NAD⁺ undergoes reactions which give rise to products that absorb light at 340 nm. These “blank” reactions hinder accurate metabolite measurements. At least two reactions are involved, a very rapid one which results in a new absorbance band maximal at 316 nm and a slow one in which an absorbance band maximal at 342 nm develops over many hours. The first of these processes is strongly pH dependent and is responsible for the high initial absorbance of metabolite assay reaction mixtures. The second reaction, which is responsible for drifting endpoints in metabolite assays, appears to be potentiated by heavy metal ions and suppressed by EDTA. It is suggested that assays involving the use of NAD⁺ in hydrazine buffers should be carried out in the presence of high concentrations of EDTA and that the pH should not be higher than is absolutely necessary to ensure a quantitative assay. The standard assays for l-lactate and l-glutamate have been substantially improved by adopting these measures.     

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK_a to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.     

Properties of a repressible alkaline phosphatase secreted by the wild-type strain 74a of neurospora crassa

A repressible extracellular alkaline phosphatase (with activity increasing steadily even up to pH 10.5) was purified from cultures of the wild-type strain 74A of Neurospora crassa, after growth on acetate and under limiting amounts of inorganic phosphate for 72 hr at 30°. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulphate (SDS). The MW was ca 172 000 and 82 000 as determined by Sephadex G-200 gel filtration and SDS-PAGE, respectively. The enzyme contained 23.6% neutral sugars, cations were not required for activity, and it was not inactivated by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) at pH 8. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of 4-nitrophenyl disodium orthophosphate (PNP-P) at pH 9 (the K_m value and Hill coefficient were 2.2 × 10^?4 M and 0.95, respectively). It was also shown that, at pH 9, the apparent number of Pi bound per dimer molecule equalled one, with a K_i value of 7.0 × 10^?4 M. The secreted enzyme showed half-lives of 23.5, 49.0 and 23.5 min at, pH 5.4, 7.4 and 9.0, respectively, after thermal inactivation at 60°. At pH 5.4, the half-life value was quite similar, while the others were respectively 2 and 4 times greater than those previously described for the repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted by ‘slime’ cells.     

An alternative 7-ethoxyresorufin o-deethylase activity assay: A continuous visible spectrophotometric method for measurement of cytochrome P-450 monooxygenase activity

A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The two assays yielded identical results with both β-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (ε₅₇₂ = 73 mm^?1 cm^?1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the K_I for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.     

Subcellular localization,metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.     

Enzymatic removal of a cephalosporin methyl ester blocking group

Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C₄ methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH₃ and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10^?10 mol min^?1 mg^?1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K_m value was 2.3 mg ml^?1. The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K₁ values of 6.63 and 0.188 mg ml^?1, respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.     

Pyruvatephosphate dikinase from Bacteroides symbiosus

1. An improved method is given for preparation of pyruvate,phosphate dikinase from Bacteroides symbiosus. 2. The bacterial enzyme is stable, free from interfering enzyme activities, and does not require thiol compounds to maintain stability during storage or assay. 3. New direct assays of enzyme activity are based on acid evolution or consumption as measured at constant pH in a pH-stat. 4. The optimum rate of reaction in the direction of pyruvate formation occurs at about pH6.4; in the direction of phosphoenolpyruvate formation, it is at pH7.2–7.8. 5. Newly determined substrate K_m values for the enzyme are: AMP, 3.5×10⁻⁶m; ATP, 1×10⁻⁴m; pyruvate, 8×10⁻⁵m; P_i, 6×10⁻⁴m. 6. K⁺ may substitute for NH₄⁺ in activating the reaction catalysed by the B. symbiosus enzyme. 7. In the direction of pyruvate formation the bivalent metal ion requirement of the enzyme is fulfilled by salts of nickel, manganese, magnesium and cobalt. In the other direction only magnesium salts were effective. 8. The nucleotide specificity of the enzyme is strictly limited to the adenine nucleotides. CTP and ITP strongly inhibit the reaction in the direction of phosphoenolpyruvate formation.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Characterization of a Novel Dye-Decolorizing Peroxidase (DyP)-Type Enzyme from Irpex lacteus and Its Application in Enzymatic Hydrolysis of Wheat Straw

Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications which presents an interesting, but still scarcely known, enzymatic oxidative system. Among these enzymes, the production, purification, and identification of a new dye-decolorizing peroxidase (DyP)-type enzyme, as well as its physico-chemical, spectroscopic, and catalytic properties, are described in the current work. According to its N-terminal sequence and peptide mass fingerprinting analyses, I. lacteus DyP showed high homology (>95%) with the hypothetical (not isolated or characterized) protein cpop21 from an unidentified species of the family Polyporaceae. The enzyme had a low optimal pH, was very stable to acid pH and temperature, and showed improved activity and stability at high H₂O₂ concentrations compared to other peroxidases. Other attractive features of I. lacteus DyP were its high catalytic efficiency oxidizing the recalcitrant anthraquinone and azo dyes assayed (k_cat/K_m of 1.6 × 10⁶ s^-1 M^-1) and its ability to oxidize nonphenolic aromatic compounds like veratryl alcohol. In addition, the effect of this DyP during the enzymatic hydrolysis of wheat straw was checked. The results suggest that I. lacteus DyP displayed a synergistic action with cellulases during the hydrolysis of wheat straw, increasing significantly the fermentable glucose recoveries from this substrate. These data show a promising biotechnological potential for this enzyme.     

Some properties of a ficin-papain inhibitor from avian egg white

A procedure has been established for the isolation, from sheep liver, of 6-phosphogluconate dehydrogenase which is homogeneous according to the criteria of the analytical ultracentrifuge, and isoelectric focusing. A systematic determination of the effects of pH, ionic strength, metal ions, and temperature, on the kinetic parameters of the isolated 6-phosphogluconate dehydrogenase has been carried out. Double-reciprocal plots of enzyme rate measurements as a function of substrate concentration indicate K_m values of 15 μm for 6-phosphogluconate, and 7 μm for NADP⁺, under optimum assay conditions, and show no effect of the concentration of one substrate on the K_m of the other substrate under the assay conditions employed. Ionic strength, monovalent and divalent metals, are apparently interchangeable in their ability to activate the enzyme, and act by decreasing the K_m values of the enzyme, not by increasing the reaction rate. Concentrations of metals above the optimum are strongly inhibitory. Plots of ?log K_m vs pH show inflection points at 8.3 for 6-phosphogluconate, and 6.5 for NADP⁺. At low substrate concentrations the pH optimum of the enzyme is at pH 7.7, but plots of V vs pH increase up to pH 9.1 (the enzyme is unstable at higher pH values). An Arrhenius plot shows a straight line between temperatures of 8.6 and 39.4 °C, and an energy of activation of 15,450 cal mole^?1.     

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

Effects of salts and pH on the rate of erythrocyte diphosphoglycerate mutase

Erythrocyte diphosphoglycerate mutase is inhibited by several inorganic salts, the extent of the effect being characteristic of the anionic component, i.e., at ionic strength of about 0.1, SO₄^2? > Cl^? > CH₃COO^?. Using a partially purified enzyme preparation from human red blood cells, kinetic constants were determined in the presence of 0.1 m KCl to simulate the ionic environment of the cell. At pH 7.5, the addition of salt caused a 10-fold increase in the K_m of 1,3-diphosphoglycerate and a 46-fold increase in the K_i of 2,3-diphosphoglycerate. There was no effect of salt on the K_m of 3-phosphoglycerate or on the maximal velocity of the reaction. In the presence of 0.1 m KCl, the _i of inorganic phosphate increased from 0.3 mm to 0.6 mm. The K_m of 1,3-diphosphoglycerate was pH dependent, the values obtained being 3.6 μm at pH 6.75, 3.1 μm at pH 7.24, and 6.7 μm at pH 7.75. The K_i values for 2,3-diphosphoglycerate under the same conditions were: 12 μm at pH 6.75, 20μm at pH 7.24, and 53 μm at pH 7.75. The relative maximal velocity of the reaction has been evaluated over the same pH range. The maximal activity of the enzyme measured at 25 °C and pH 7.5 was 2 units/min/ml of packed red cells. From these studies, it is concluded that the effective enzymatic rate increases fourfold when the pH increases from 6.75 to 7.75.     

Catalysis of hydrolysis and aminolysis of benzylpenicillin mediated by a ternary complex with zinc ion and Tris(hydroxymethyl)aminomethane

It was discovered that the combination of zinc ion and Tris in the pH range 7.5–10 is a very effective true catalyst for hydrolysis and aminolysis (by Tris) of benzylpenicillin. Both Cu²⁺ and Ni²⁺ ions were nonreactive in this system. The rate of loss of penicillin from solution was found to be a linear function of zinc ion at concentrations up to 10^?5M, while the dependence upon Tris concentration is maximal at about 0.02–0.03 M at each pH studied. At high penicillin concentrations saturation kinetics was observed. Product assays showed the major product to be a penicilloic acid at low Tris concentrations and N(penicilloyl)-Tris at high Tris concentrations. A mechanism is proposed which suggests that the reaction is mediated by a ternary complex in which the metal ion acts to bring the reactants (penicillin and Tris) into close proximity and to lower the pK_a of a Tris hydroxyl, creating a strong nucleophile. This mechanism also explains the results of the product assays and the lack of reactivity of the other metal ions tested. This reaction may be related to the mechanism for a known zinc-dependent β-lactamase.     

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.