The study of hydrolysis of new lipid-like substrates and trilaurin in monolayers catalyzed with the lipase from Pseudomonas fluorescens

A simple method for determining the enzymic hydrolysis parameters of lipid-like substrates and trilaurin assembled in monolayers at the water-air interface was suggested. At a surface pressure of 10 mN/m, the initial rates of lipolysis were found to be proportional to the decrease in area of the substrate monolayer caused by the enzymic hydrolysis in a single-compartment Langmuir balance. The kinetic parameters for the hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acetylated in position 2 with an amino acid (phenylalanine, leucine, or valine) catalyzed with lipase from Pseudomonas fluorescens were determined. Unlike models of enzymic hydrolysis that neglect the thickness of the substrate monolayer, our method allows the determination of kinetic parameters in standard dimensions. The values of kcat for the synthetic pseudoglycerides were found to be significantly higher than that for trilaurin, while the values of Km(app) were close. This may be due to the presence of positively charged primary amino groups in the molecules of pseudoglycerides.     

Non-Productive Binding of Substrates as One of Aspects of Substrate Specificity of Cholinesterases of Different Origin

Taking into account the phenomenon of non-productive binding of substrate, kinetic parameters of hydrolysis of acetylcholine (ACh) and its 13 derivatives with different structures of ammonium group by cholinesterase (ChE) of human erythrocytes, ChE of horse blood serum, and ChE of optic ganglia of the Pacific squid Todarodes pacificus are determined. A dependence is revealed of values of parameters of their enzymatic hydrolysis and parameters of the non-productive binding on the substrate structure and ChE nature. Effects of salts, LiCl, NaCl, KCl, MgCl₂, CaCl₂ and BaCl₂, on various kinetic parameters, including parameters of the non-productive binding of substrate, of enzymatic hydrolysis of iodides of ACh and N-acetoxyethylene-N-ethylpiperidinium under action of horse blood serum ChE are studied. Addition of the salts to the reaction mixture produced different effects on values of the catalytic center activity (a _c) and the Michaelis constant (K _M), depending on the cation nature and the substrate structure. At the same time, values of the a _c/K _M ratio that characterize to a degree the substrate affinity to the enzyme are equal to each other for two substrates differing in structure, regardless of the presence and nature of the studied cations. Parameters of the non-productive binding of N-acetoxyethylene-N-ethylpiperidinium iodide also depended on the salt nature; however, in that case, a question arises as to the correctness of the comparative analysis, when at determinations of the parameters the non-productive binding of ACh is ignored.     

Some Properties of Endo-polygalacturonase from Trichosporon penicillatum SNO-3

Some properties of the endo-polygalacturonase from Trichosporon penicillatum were investigated. The enzyme showed the highest activity around pH 5.0 and was stable at this pH up to 50°C. The enzyme catalyzed the hydrolysis of galacturonic acid oligomers as well as its polymer. The pentamer was degraded to a trimer and a dimer, the tetramer to a trimer and a monomer, and the trimer to a dimer and a monomer, respectively, whereas the dimer was not degraded. The kinetic constant V_max and Km values changed with the substrate chain-length; the Km values tended to decrease, whereas the V_max values tended to increase with increasing chain-length of the substrate. The amino acid residue participating in the active site of the enzyme was studied and it was found to be histidine.     

On the question of an electrokinetic requirement for phospholipase C action

Summary The phospholipase C ofclostridium welchii ( toxin) has an absolute requirement for trace quantities of Ca²⁺. It attacks pure phosphatidylcholine particles (smectic mesophases) having a close-packed bilayer structure only when their surface zeta potential is made positive by the addition of certain divalent ions (e.g., Ca²⁺) to the aqueous phase or by the presence of low concentrations of long chain cations to the lipid. Alternatively, if the rotational freedom of individual phospholipid molecules is increased by the insertion of shortn-alkanols (e.g., hexanol) into the bilayer or when a monolayer of the substrate at an air/water interface is expanded, enzymic hydrolysis can occur without any requirement for a net positive charge on the surface.     

A substrate channel in the nitrogenase MoFe protein

Nitrogenase catalyzes the six electron/six proton reduction of N₂ to two ammonia molecules at a complex organometallocluster called “FeMo cofactor.” This cofactor is buried within the α-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the reduction of the substrates N₂, acetylene, azide, and propyne. For each MoFe protein, V _max values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the K _m values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.     

Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/izooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of palm oil in a lecithin/isooctane reversed micellar system. The reaction obeys Michaelis-Menten kinetics for the initial conditions. Kinetic parameters such as maximum rate and Michaelis constant (K _m) were determined for lipase-catalyzed hydrolysis in n-hexane and isooctane. According to the K _m values, the enzyme affinity towards the substrate was increased in isooctane. The maximum degree of hydrolysis was generally decreased as the initial substrate concentration was increased. This may suggest that the hydrolysis in lecithin reversed micelles should be regarded as a one-substrate first-order reversible reaction. It is shown in this study that the proposed one-substrate first-order kinetic model can serve for the precise prediction of the degree of hydrolysis for a known reaction time or vice versa, when the initial substrate concentration is less than 0.325 mol/dm³. A disagreement with this model was found when the initial substrate concentration was higher than approximately 0.3 mol/dm³. This may be due to the effects of the products on lipase activity or even to the conversion of the reversed micellar system to other systems. Received: 16 May 1997 / Received revision: 22 October 1997 / Accepted: 24 October 1997     

Kinetics of enzymic hydrolysis of malto-oligosaccharides: A comparison with acid hydrolysis

The hydrolysis of malto-oligosaccharides G₃-G₆ catalysed by porcine pancreatic alpha-amylase was investigated kinetically at 25°. Kinetic parameters corresponding to different positions of enzymic attack were determined and product inhibition was evaluated. The enzymic hydrolysis was compared in terms of reaction rate and pattern of action with hydrolysis in 0.1m H₂SO₄ at 70°. Mathematical models for the mechanism of hydrolysis were developed and a good rationalisation of the experimental results was achieved.     

New role of C-terminal 30 amino acids on the insoluble chitin hydrolysis in actively engineered chitinase from <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments, respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His–Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular mass of 65,713 and 62,723 Da, respectively. The results of biochemical characterization including kinetic parameters, spectroscopy of fluorescence and circular dichroism, chitin-binding and hydrolysis, and thermostability, both VpChiA and VpChiAG589, had very similar physicochemical properties such as the optimum pH (6), temperature (40°C), and kinetic parameters of Km and kcat against the 4MU–(GlcNAc)₂ or 4MU–(GlcNAc)₃ soluble substrates. The significant increase of thermostability and the drastic decrease of the hydrolyzing ability of VpChiAG589 toward the insoluble α-chitin substrate suggested that a new role could be played by the C-terminal 30 amino acids.     

Simultaneous hydrolysis of tri- and tetrasaccharides by industrial mixtures of glucoamylase and α-amylase: kinetics and thermodynamics

The present paper deals with the study of the kinetics and thermodynamics of batch enzymic hydrolysis of multisubstrate media in the presence of more than one glucanase. A kinetic model, similar to the one proposed for competitive inhibition, is presented to describe the competition between two substrates, tri- and tetrasaccharides, for the same enzyme, glucoamylase. A literature research on the most recurrent values of the Michaelis-Menten constant also shows a linear relationship between this parameter and the molecular weight of the sugar substrate for a given glucanase.List of Symbols a dimensionless empirical parameter in Eq. (1) - b dimensionless empirical parameter in Eq. (1) - B dimensionless empirical parameter in Eq. (6) - E kg/m³ enzyme concentration - E _a kcal/mol activation energy - kc kg/m³ competition constant - k _M kg/m³ Michaelis-Menten constant - MW kg·10^–3 molecular weight - r mol/(min·kg) specific hydrolysis rate of glucoamylase - R cal/(°K·mol) ideal gas constant - S kg/m³ substrate concentration - T °K absolute temperature - v kg/(m³·h) hydrolysis rate of glucoamylase Indices 1 values referring to trisaccharides - 2 values referring to tetrasaccharides - 0 starting values - max maximum values     

Conclusions about aminopeptidase in tissue sections from studies of amino acid naphthylamide hydrolysis

Summary Catalytic properties (K_M, V_max) of aminopeptidase in pig kidney sections, in isolated membranes and in a solubilized purified form were investigated using amino acid 2-naphthylamides and 4-methoxy-2-naphthylamides. In the first case these properties were estimated on the basis of the stain intensity resulting from the coupling of product with Fast Blue B, in the latter two cases they were measured fluorometrically. The following observations were made: (1) In all three cases the substrate turnover was shown to be a direct function of time and enzyme concentration. (2) The values obtained for the solubilized and the membrane bound form were practically identical but differed from those found in tissue sections. (3) Each amino acid derivative had defined constants, but these were difficult to obtain in sections, especially if it was necessary, on account of poor solubilities, to use low substrate concentrations. (4) Hydrophilic amino acid derivatives were adsorbed to tissue membranes much less than hydrophobic ones. (5) Fast Blue B caused a non-competitive inhibition of enzymic activity. (6) Binding of antibody against pure aminopeptidase caused inhibition of the enzymic hydrolysis of all the naphthylamides. Thus, histochemical stain intensities per time and area derived from one substrate at a defined concentration are suitable for the determination of enzyme concentrations. However, no conclusions regarding the homogeneity of the enzyme in sections can be drawn by comparing the stain intensities obtained with different substrates in contrast to data from the inhibition of substrate hydrolysis by antibody.     

Simultaneous estimation ofV max,Km, and the rate of endogenous substrate production (R) from substrate depletion data

The nonlinear and 3 linearized forms of the integrated Michaelis-Menten equation were evaluated for their ability to provide reliable estimates of uptake kinetic parameters, when the initial substrate concentration (S₀) is not error-free. Of the 3 linearized forms, the one where t/(S₀–S) is regressed against ln(S₀/S)/(S₀–S) gave estimates ofV _max and K_m closest to the true population means of these parameters. Further, this linearization was the least sensitive of the 3 to errors (±1%) in S₀. Our results illustrate the danger of relying on r² values for choosing among the 3 linearized forms of the integrated Michaelis-Menten equation. Nonlinear regression analysis of progress curve data, when S₀ is not free of error, was superior to even the best of the 3 linearized forms. The integrated Michaelis-Menten equation should not be used to estimateV _max and K_m when substrate production occurs concomitant with consumption of added substrate. We propose the use of a new equation for estimation of these parameters along with a parameter describing endogenous substrate production (R) for kinetic studies done with samples from natural habitats, in which the substrate of interest is an intermediate. The application of this new equation was illustrated for both simulated data and previously obtained H₂ depletion data. The only means by whichV _max, K_m, and R may be evaluated from progress curve data using this new equation is via nonlinear regression, since a linearized form of this equation could not be derived. Mathematical components of computer programs written for fitting data to either of the above nonlinear models using nonlinear least squares analysis are presented.     

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.     

Extracellular phosphatase activity in sediments of the Breitenbach,a Central European mountain stream

Activity of extracellular phosphatases (phosphomonoesterases) was measured in sandy streambed sediments of the Breitenbach, a small unpolluted upland stream in Central Germany. Fluorigenic 4-methylumbelliferyl phosphate served as a model substrate. Experiments were conducted using sediment cores in a laboratory simulation of diffuse groundwater discharge through the stream bed, a natural process occurring in the Breitenbach as well as many other streams.Streambed sediments contained high levels of particulate phosphorus, but concentrations of dissolved phosphorus in the interstitial water were 3 to 4 orders of magnitude lower. These interstitial concentrations were similar to those in the stream and groundwater. Extracellular phosphatase activity was high in the streambed sediments. These enzymes probably contribute significantly to the flux of phosphorus in sediment by hydrolyzing phosphomonoesters, making free phosphate available to the sediment microorganisms.Factors influencing the kinetic parameters V _max (maximum activity) and apparent K _m (enzyme affinity) of phosphatase were discharge rates of water through the sediment, water quality (ground- or stream water), and substrate (phosphomonoesters) as well as dissolved ortho-phosphate concentrations. Enzymes are supposed to be effective at limiting substrate concentrations, where, in this study, changes in discharge rates had little influence on rates of hydrolysis. Higher V _max and lower K _m values were found during percolation of groundwater through the sediment cores, compared with stream water. This indicates that rates of hydrolysis were higher with groundwater, both at substrate limitation and at substrate saturation. This was probably a consequence of the lower levels of dissolved ortho-phosphate in the groundwater.     

Kinetic Behaviour of Free Lipase and Mica-Based Immobilized Lipase Catalyzing the Synthesis of Sugar Esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K _m and V _max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K _m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V _max,app>V _max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Facilitated diffusion with consecutive reaction: optimal carrier affinity

The interplay between facilitated diffusion of a substrate through a membrane and a consecutive enzymic reaction, both of which follow Michaelis-Menten kinetics, has been theoretically investigated and the effect of the kinetic and transport parameters on the rate of substrate uptake is graphically illustrated. At steady state two characteristic features of the system have been identified. First, the substrate concentration at the internal enzymic side of the membrane cannot exceed a given value even at much higher external substrate concentrations. Second, the uptake rate is maximum at a given value of K_T, the kinetic parameter of the transport system that expresses the reciprocal carrier affinity of the substrate. The optimum value of K_T is approximately equal to the external substrate concentration. This particular dependence of the uptake rate on the carrier affinity is expected to play an important role in hormonal regulation.     

Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k_cat = 0.13 ± 0.02 min ? ¹ and K_s = 0.67 ± 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k_cat = k₂). Though the aryl acylamidase activity of albumin is low (k_cat/K_s = 195 M^{? 1}min^{? 1}), because of its high concentration in human plasma (0.6–1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.     

A kinetic correlation for konjac powder hydrolysis by β-mannanase from Bacillus licheniformis

The kinetics of konjac powder hydrolysis by -mannanase from Bacillus licheniformis was studied for the production of manno-oligosaccharides, a bifidobacteria factor. It is assumed that, during the whole reaction, the substrate has an average degree of polymerization with which the apparent kinetic constants, K _m and k ₊₂, vary and the concentration of substrate remains unchanged. Based on the above assumptions, a set of simple expressions is proposed to correlate the initial concentration of substrate, enzyme concentration, degree of hydrolysis and reaction time. The results predicted are in good agreement with the experimental data.     

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimirystoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18–28°C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots ofK _m also show breaks in the same temperature interval, with the exception of the dioleoylenzyme vesicles, whereK _m is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24°C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3–18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.     

Quantitative assessment of primary cerium reaction product formed by glucose-6-phosphatase activity in a male rat liver: an image analysis study

 Glucose-6-phosphatase (G6Pase) activity has been determined in periportal and pericentral areas of the liver of normal male rats. Measurements were performed on unfixed cryostat sections mounted on semipermeable membranes. In the present study, the oxidized primary reaction product of a cerium-based histochemical method [Ce(IV)perhydroxyphosphate] instead of the final reaction product after a second-step incubation was measured. For quantification of the amount of Ce(IV)perhydroxyphosphate formed the digital image analyzing system Quantimet 500+ was used. Estimated values of optical densities of Ce(IV)perhydroxyphosphate over test areas were employed for calculation of kinetic parameters of (G6Pase). Highest activities of G6Pase (higher K _m and V _max levels) were found in periportal areas of the rat liver, indicating a higher amount of active enzyme molecules and a lower affinity for the substrate. Differences in values for both K _m and V _max between periportal and pericentral zones were highly significant and closely comparable to those for male fed rats. Correlations between K _m and V _max were significant for periportal as well for pericentral liver areas. The results of the present study thus allow the same biological implications as histochemical methods employing a final reaction for quantification of enzyme activities. The present method avoids the drawbacks of enhancement reactions and demonstrates the feasibility of in situ analysis of enzyme kinetic parameters by quantification of oxidized primary cerium reaction products. Accepted: 8 January 1996     

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.