Different kinetic patterns in the alpha-chymotrypsin-catalysed hydrolysis of synthetic ester substrates

The reaction of alpha-chymotrypsin with AcTyr-OEt and with AcTrp-OEt at pH 7.0 and 7.8 was studied over a wide range of substrate concentrations. The reaction with AcTyr-OEt at pH 7.8 was shown to be non-hyperbolic using a variety of criteria whereas those at pH 7.0 with the same substrate and at both pH values with AcTrp-OEt were hyperbolic. The non-hyperbolicity of the reaction with AcTyr-OEt at pH 7.8 followed a pattern of negative cooperativity with a Hill coefficient for the high substrate concentration range of 0.48. Although other explanations are possible, the pH dependence of the reaction with AcTyr-OEt could be related to the slow transition of the two known forms of the enzyme.     

Facile synthesis of glucose-1-phosphate from starch by Thermus caldophilus GK24 α-glucan phosphorylase

The glgP gene encoding α-glucan phosphorylase (α-GP) from the thermopile Thermus caldophilus GK24 has been identified, cloned, and overexpressed in Escherichia coli and used to synthesize d-glucose-1-phospate (G1P) from an inexpensive starch. The enzyme, purified 6.5-fold, was isolated in 31% yield from the transformed E. coli, and gave a single band. The purified enzyme may exist as a homohexamer with an apparent molecular mass of a 550 kDa molecule, consisting of 90 kDa per subunit. The optimal pH and temperature were 7.0 and 70 °C in the α-GP reaction with starch producing G1P. Soluble starch (amylopectin, amylose) turned out to be a better substrate giving a higher yield of G1P than α-1,6-branched α-1,4-glucans (glycogen, potato starch, etc.). As a result, G1P was obtained in a good yield (47%, w/w) from the reaction containing 5% (w/v) soluble starch in 0.7 M potassium phosphate at pH 7.0. T. caldophilus α-GP shows a high tolerance (up to 0.7 M) of potassium phosphate and plays a critical role in shifting the reaction equilibrium in favor of G1P synthesis. The G1P product can be purified simply by ethanol precipitation, after removing the unreacted starch and inorganic phosphate by activated charcoal and magnesium acetate precipitation. It is concluded that T. caldophilus α-GP readily utilized in large scale synthesis of G1P.     

Mechanisms of the acido- and thermo-phily of Cyanidium caldarium Geitler I. Effects of temperature, pH and light intensity on the photosynthetic oxygen evolution of intact and treated cells

Photosynthetic oxygen evolution in an acido- and thermo-philicunicellular alga, Cyanidium caldarium, was measured under variousconditions, using a Clark-type oxygen electrode. 1). Maximum Hill reaction activity with p-benzoquinone as theHill oxidant was obtained at 45°C in a wide pH range from1.0 to 7.0. 2) The pH activity curve showed two peaks at pH3.0 and 7.0. The Hill activity had an optimum at pH 3.0 in cellspreilluminated under strong light (300,000 lux, 30 min, 40°C).Sonication of algal cells abolished the pH 3.0 component ofthe Hill reaction producing an activity maximum at pH 7.0. 3)Endogenous O₂ evolution in the absence of the Hill oxidant,which lasted for several minutes after illumination, had a maximumat pH 7.0. 4) This endogenous O₂ evolution was abolished bysonication. 5) KCN inhibited endogenous O₂ evolution, but notthe Hill reaction in the presence of p-benzoquinone. (Received August 19, 1974; )     

In vitro evaluation of feed-grade enzyme activity at pH levels simulating various parts of the avian digestive tract

The activities of β-glucanase, xylanase, amylase, α-galactosidase and protease were measured at their published optimum pH levels and at pH levels of 3.0, 6.0, 6.5, 7.0 and 7.5 to simulate pH levels of the gizzard, the diet, the crop, and the proximal and distal parts of small intestine, respectively. The activity of β-glucanase was determined by measuring reducing sugars after incubation of β-glucan. Xylanase activity was assayed by measuring xylose after hydrolysis of xylan. The activity of amylase was measured through hydrolysis of soluble starch. The assay of α-galactosidase was based on a hydrolysis of p-nitrophenyl-α-d-galactoside followed by measurement of liberated p-nitrophenol. The activity of protease was assayed by measuring tyrosine after enzymatic hydrolysis of casein. β-Glucanase had high activity at pH levels of 3.0–7.0. Xylanase had no enzyme activity at pH 3.0, but had high activity at pH levels of 6.0–7.0. Amylase had high activity at pH levels of 6.0 and 6.5 but had no or very low activity at pH 3.0, 7.0 and 7.5. α-Galactosidase had high activity at pH 6, but not at other pH levels tested. Protease had either no or very low activity at all pH levels except at pH 3.0. These results suggest that the pH levels commonly found in the avian digestive tract may be a limiting factor for maximum activity of the exogenous enzymes, such as amylase, α-galactosidase and protease.     

Bovine intermediate pituitary α-amidation enzyme: Preliminary characterization

A secretory granule-associated enzymatic activity that converts mono-[¹²⁵I]-D-Tyr-Val-Gly into mono-[¹²⁵I]-D-Tyr-Val-NH₂ has been studied. The activity is primarily soluble and shows optimal activity at pH 7 to pH 8. Amidation activity was stimulated 9-fold by addition of optimal amounts of copper (3 μM). In the presence of optimal copper, ascorbate stimulated the reaction 7-fold; none of the other reduced or oxidized cofactors tested was as effective. Taking into account the dependence of the reaction on ascorbate and molecular oxygen and the production of glyoxylate [2], it is suggested that the α-amidation enzyme is a monooxygenase. Lineweaver Burk plots with D-Tyr-Val-Gly as the varied substrate demonstrated Michelis-Menten type kinetics with the values of K_m and V_max increasing with the addition of ascorbate to the assay. A variety of peptides ending with a COOH-terminal Gly residue act as inhibitors of the reaction. Two synthetic peptides, γ₂MSH and ACTH(1–14), with carboxyl termini similar to the presumed physiological substrates for the enzyme, act as competitive inhibitors with similar K₁ values. It is likely that this secretory granule α-amidation activity is involved in the physiological biosynthetic α-amidation of a wide range of bioactive peptides.     

Some kinetic properties of the β- -glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

In situ behaviour of D(-)-lactate dehydrogenase from Escherichia coli

Some kinetic properties of the D(-)-lactate dehydrogenase (EC 1.1.1.28) of Escherichia coli have been investigated. There were marked differences between the kinetic properties of the enzyme studied in situ compared with the in vitro D(-)-lactate dehydrogenase. D(-)-Lactate dehydrogenase in situ showed high substrate inhibition with pyruvate over the pH range 6.0–7.0, whereas the enzyme in vitro did not. The pH optimum for pyruvate reduction by the in situ D(-)-lactate dehydrogenase ranged between pH 7.5 and 7.8, whereas the in vitro enzyme showed its pH optimum between pH 6.8 and 7.0. The pK values of the prototropic groups that controlled the enzymatic activity shift to the acidic region for the in vitro enzyme with respect to the in situ enzyme. In vitro D(-)-lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate and its coenzyme, NADH, at pH values ranging between pH 6.0 and 8.5, but the in situ enzyme showed homotropic interactions neither with pyruvate nor with NADH at all pH values studied.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Conformational change of poly( -lysine) induced by lipid vesicles of dilauroylphosphatidic acid

The effect of negatively charged dilauroylphosphatidic acid (DLPA) vesicles on the conformation of poly( -lysine) was investigated by circular dichroism measurements. DLPA vesicles induced a confomiational change Of poly( -lysine) from the random coil to β-structure in 5 mM Tes, pH 7.0. The fraction of induced β-structure (F_β) was determined via a procedure of curve fit the observed spectra to the reference spectra. F_β increased linearly with the molar ratio, r, of DLPA to lysine residues up to r 0.7, and reached a saturation value of 1 at r > 1. Within the range 0.7 r 1, precipitation occurred. The effect of dilution of the negative charge on vesicle membranes was examined by mixing DLPA with dilauroylphosphatidylcholine (DLPC). Although the β-structure Of poly -lysine) was also induced by mixed vesicles, the saturation value of F_β decreased with decreasing DLPA content in mixed vesicles. The variation in saturation value of F_β with the composition of mixed vesicles was interpreted in terms of the change in average distance between DLPA head groups in mixed vesicles.     

Phosphate transport in human red blood cells: Concentration dependence and pH dependence of the unidirectional phosphate flux at equilibrium conditions

Summary The concentration dependence and the pH dependence of the phosphate transport across the red cell membrane were investigated. The unidirectional phosphate fluxes were determined by measuring the³²P-phosphate self-exchange in amphotericin B (5 mol/liter) treated erythrocytes at 25°C.The flux/concentration curves display anS-shaped increase at low phosphate concentrations, a concentration optimum in the range of 150 to 200mm phosphate and a self-inhibition at high phosphate concentrations. The apparent half-saturation concentrations,P _(0.5), range from 50 to 70mm and are little affected by pH. The self-inhibition constants, as far as they can be estimated, range from 400 to 600mm. The observed maximal phosphate fluxes exhibit a strong pH dependence. At pH 7.2, the actual maximal flux is 2.1×10^–6 moles·min^–1·g cells^–1. The ascending branches of the flux/concentration curves were fitted to the Hill equation. The apparent Hill coefficients were always in the range of 1.5–2.0. The descending branches of the flux/concentration curves appear to follow the same pattern of concentration response.The flux/pH curves were bell-shaped and symmetric with regard to their pH dependence. The pH optimum is at approximately pH 6.5–6.7. The apparent pK of the activator site is in the range of 7.0 to 7.2, while the apparent pK for the inactivating site is in the range of 6.2 to 6.5. The pK-values were not appreciably affected by the phosphate concentration.According to our studies, the transport system possesses two transport sites and probably two modifier sites as indicated by the apparent Hill coefficients. In addition, the transport system has two proton binding sites, one with a higher pK that activates and one with a lower pK that inactivates the transport system. Since our experiments were executed under self-exchange conditions, they do not provide any information concerning the location of these sites at the membrane surfaces.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Investigation of the active site of the extracellular β-<Emphasis Type="SmallCaps">D</Emphasis>-glucosidase from <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis>

Summary The catalytic amino acid residues of the extracellular β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus carbonarius were investigated. The pH dependence curves gave apparent pK values of 2.8 and 5.93 for the free enzyme, and 2.24 and 6.14 for the enzyme–substrate complex using p-nitrophenyl-β-D-glucoside as substrate. Carbodiimide- and Woodward reagent K-mediated chemical modifications suggested that a carboxylate residue, located in the active centre, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.61 in the modification reaction, proving that a carboxylate residue was modified. The A. carbonarius β-glucosidase was irreversibly inactivated by N-bromoacetyl-β-D-glucopyranosylamine. The active site specificity of the inactivation was proved by using the competitive inhibitor p-nitrophenyl-1-thio-β-D-glucopyranoside. pH Dependence studies of inactivation revealed that modification by N-bromoacetyl-β-D-glucopyranosylamine could be directed toward the carboxylate group acting as the catalytic nucleophile, as in the case of the carbodiimide and Woodward reagent K modifications.     

Structural insights into the mechanism of pH-selective substrate specificity of the polysaccharide lyase Smlt1473

Polysaccharide lyases (PLs) are a broad class of microbial enzymes that degrade anionic polysaccharides. Equally broad diversity in their polysaccharide substrates has attracted interest in biotechnological applications such as biomass conversion to value-added chemicals and microbial biofilm removal. Unlike other PLs, Smlt1473 present in the clinically relevant Stenotrophomonas maltophilia strain K279a demonstrates a wide range of pH-dependent substrate specificities toward multiple, diverse polysaccharides: hyaluronic acid (pH 5.0), poly-β-D-glucuronic (celluronic) acid (pH 7.0), poly-β-D-mannuronic acid, and poly-α-L-guluronate (pH 9.0). To decode the pH-driven multiple substrate specificities and selectivity in this single enzyme, we present the X-ray structures of Smlt1473 determined at multiple pH values in apo and mannuronate-bound states as well as the tetra-hyaluronate-docked structure. Our results indicate that structural flexibility in the binding site and N-terminal loop coupled with specific substrate stereochemistry facilitates distinct modes of entry for substrates having diverse charge densities and chemical structures. Our structural analyses of wild-type apo structures solved at different pH values (5.0–9.0) and pH-trapped (5.0 and 7.0) catalytically relevant wild-type mannuronate complexes (1) indicate that pH modulates the catalytic microenvironment for guiding structurally and chemically diverse polysaccharide substrates, (2) further establish that molecular-level fluctuation in the enzyme catalytic tunnel is preconfigured, and (3) suggest that pH modulates fluctuations resulting in optimal substrate binding and cleavage. Furthermore, our results provide key insight into how strategies to reengineer both flexible loop and regions distal to the active site could be developed to target new and diverse substrates in a wide range of applications.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.     

Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

Culture requirements for the production of protease by Aspergillus oryzae in solid state fermentation

Summary A number of culture conditions for protease production by Aspergillus oryzae NRRL 2160 on solid substrates were investigated. The pH of the medium and the substrate markedly affected protease production. High protease yield was obtained when the fungus was cultivated for 72–96 h on rice hulls: rice bran (7:3), at an initial pH of 7.0. Maximal protease production was achieved at an initial moisture content of 35–40%, corresponding to a water activity range of 0.982–0.986. Casein and gluten were effective inducers. Polyethylene bags proved to be promising containment systems for solid state cultivation. Offprint requests to: A. M. R. Pilosof     

Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia

In this study we purified and characterized a fibrinolytic protease from the mycelia of Perenniporia fraxinea. The apparent molecular mass of the purified enzyme was estimated to be 42 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), fibrin zymography and size exclusion using fast protein liquid chromatography (FPLC). The first 20 amino acid residues of the N-terminal sequence were ASYRVLPITKELLPPEFFVA, which shows a high degree of similarity with a fungalysin metallopeptidase from Coprinopsis cinerea. The optimal reaction pH value and temperature were pH 6.0 and 35–40 °C, respectively. Results for the fibrinolysis pattern showed that the protease rapidly hydrolyzed the fibrin α-chain followed by the β-chain. The γ–γ chains were also hydrolyzed, but more slowly. The purified protease effectively hydrolyzed fibrinogen, preferentially digesting the Aα-chains of fibrinogen, followed by Bβ- and γ-chains. We found that protease activity was inhibited by Cu²⁺, Fe³⁺, and Zn²⁺, but enhanced by the additions of Mn²⁺, Mg²⁺ and Ca²⁺ metal ions. Furthermore, the protease activity was inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The mycelia of P. fraxinea may thus represent a source of new therapeutic agents to treat thrombosis.     

The effect of α-factor on the rate of cell-cycle initiation in Saccharomyces cerevisiae : α-Factor modulates transition probability in yeast

The rate of cell-cycle initiation was studied in a-cells of S. cerevisiae in the presence of the synthetic analogue of α-factor [N-Trp, Arg⁷]-α-factor (TA-αF). It was shown that TA-αF lowers the rate constant of cell-cycle initiation (or transition probability) for each separate cell. It was concluded on the basis of these results that the term ‘arrest’ in G1 by α-factor should be interpreted in a quantitative sense as the decrease in the probability of the emergence from ‘start’ per unit time and not as being equivalent to an ‘all-or-none’ response. The dependence of the rate constant of the cell-cycle initiation on the concentration of TA-αF can be described by the Hill equation where n = 1.01 + 0.05 and K = 14.5 ± 2.5 nM (±S.E.). It is demonstrated that after transfer of cells into the medium with a higher or a lower concentration of TA-αF, the rate constant of cell-cycle initiation changes abruptly from one value to another after a lag-period of 30 and 40 min respectively. This suggests a multistep mechanism of action for α-factor. The difference in the lag-periods allows us to suggest that α-factor exerts its action by two independent pathways. Since the Hill coefficient is practically equal to unit, no cooperative interactions are likely to be involved at least in one of these pathways. The inhibition of cell-cycle initiation can be used as a more adequate and sensitive test for biological activity of α-factor as compared to morphological measurements.     

The Importance of pH in Regulating the Function of the Fasciola hepatica Cathepsin L1 Cysteine Protease

The helminth parasite Fasciola hepatica secretes cathepsin L cysteine proteases to invade its host, migrate through tissues and digest haemoglobin, its main source of amino acids. Here we investigated the importance of pH in regulating the activity and functions of the major cathepsin L protease FheCL1. The slightly acidic pH of the parasite gut facilitates the auto-catalytic activation of FheCL1 from its inactive proFheCL1 zymogen; this process was ∼40-fold faster at pH 4.5 than at pH 7.0. Active mature FheCL1 is very stable at acidic and neutral conditions (the enzyme retained ∼45% activity when incubated at 37°C and pH 4.5 for 10 days) and displayed a broad pH range for activity peptide substrates and the protein ovalbumin, peaking between pH 5.5 and pH 7.0. This pH profile likely reflects the need for FheCL1 to function both in the parasite gut and in the host tissues. FheCL1, however, could not cleave its natural substrate Hb in the pH range pH 5.5 and pH 7.0; digestion occurred only at pH≤4.5, which coincided with pH-induced dissociation of the Hb tetramer. Our studies indicate that the acidic pH of the parasite relaxes the Hb structure, making it susceptible to proteolysis by FheCL1. This process is enhanced by glutathione (GSH), the main reducing agent contained in red blood cells. Using mass spectrometry, we show that FheCL1 can degrade Hb to small peptides, predominantly of 4–14 residues, but cannot release free amino acids. Therefore, we suggest that Hb degradation is not completed in the gut lumen but that the resulting peptides are absorbed by the gut epithelial cells for further processing by intracellular di- and amino-peptidases to free amino acids that are distributed through the parasite tissue for protein anabolism.