Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A₂ (PLA₂) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA₂ toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and d-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA₂ and most of the hydrolysis products desorb from substrate surface in 40 min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA₂ increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA₂ preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA₂-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA₂ mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA₂.     

Variables Affecting Two Electron Transport System Assays

Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methanol was unsatisfactory. Phosphate buffer (0.06 M) produced higher activity than did either U.S. Environmental Protection Agency reconstituted hard water or Tris buffer sediment diluents. Intracellular formazan crystals were dissolved within minutes when in contact with immersion oil. Greater crystal production (respiration) detected by a tetrazolium salt assay occurred at increased substrate concentrations. Test diluents containing macrophyte exudates produced greater activity than did phosphate buffer, U.S. Environmental Protection Agency water, or ultrapure water diluents. Both assays showed decreases in sediment or bacterial activity through time.     

Calcium and magnesium dependence of phospholipase A2-catalyzed hydrolysis of phosphatidylcholine small unilamellar vesicles.

The Ca2+ requirement for lipid hydrolysis catalyzed by phospholipase A2 from Agkistrodon piscivorus piscivorus (App-D49) and porcine pancreas has been examined using small, unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV). Hydrolysis was affected by product inhibition even at early times, and the extent of this inhibition depended on the concentration of divalent cations. The Ca2+ requirement for half-maximal rates of hydrolysis reflected, in part, this non-catalytic role of divalent cations. The presence of 10 mM Mg2+, a cation which does not support catalysis, reduced the Ca2+ required for half-maximal rates of hydrolysis from millimolar concentrations to 40 microM for App-D49. Since the dissociation constant of the enzyme for Ca2+ in solution is 2 mM, these results indicate a change in the interaction of the enzyme with Ca2+ under catalytic conditions. The kinetic dissociation constant of Ca2+ for the pancreatic enzyme was 20 microM which is substantially lower than the dissociation constant in solution, 0.35 mM. The similarity of apparent kinetic dissociation constants for these enzymes suggests that structurally similar features determine the affinity for Ca2+ under catalytic conditions. Evidence is presented that the affinity of phospholipase A2 for Ca2+ changes subsequent to the initial interaction of the enzyme with the substrate interface. However, the apparent Michaelis constant, KMapp, for App-D49, 0.03-0.06 mM, is independent of [Ca2+] and is about the same as the equilibrium dissociation constant for DPPC SUV, 0.14 mM. We thus suggest that KMapp is a steady-state constant.     

All-in-one assay for β-d-galactoside sialyltransferases: Quantification of productive turnover,error hydrolysis,and site selectivity

Sialyltransferases are important enzymes of glycobiology and the related biotechnologies. The development of sialyltransferases calls for access to quick, inexpensive, and robust analytical tools. We have established an assay for simultaneous characterization of sialyltransferase activity, error hydrolysis, and site selectivity. The described assay does not require expensive substrates, is very sensitive (limit of detection = 0.3 μU), and is easy to perform. It is based on sialylation of nitrophenyl galactosides; the products thereof are separated and quantified by ion pair reversed phase high-performance liquid chromatography with ultraviolet detection.     

A monolayer and bulk study on the kinetic behavior of Pseudomonas glumae lipase using synthetic pseudoglycerides

A heat-stable lipase from Pseudomonas glumae was purified to homogeneity. Its positional and stereospecific properties were investigated and compared with those of the well-known porcine pancreatic lipase. The kinetic properties of both enzymes were determined by use of six isomeric synthetic pseudoglycerides all composed of a single hydrolyzable fatty acyl ester bond and two lipase-resistant groups: one acylamino and one ether function. Two enzyme assay techniques were applied: a detergent-free system, the monomolecular surface film technique, and the pH-stat technique using clear micellar solutions of substrate in the presence of Triton X-100. Regarding the cleavage of primary ester bonds, P. glumae lipase possesses no stereopreference. In contrast, a large stereopreference in favor of the R-isomer is found for the hydrolysis of secondary ester bonds. Secondary ester bonds are efficiently cleaved by the lipase, which makes it of potential interest for enzymatic synthetic purposes. For the hydrolysis of this R-isomer a correlation between the experimental catalytic turnover rate and the binding constant for micelles was observed. The kinetic data of P. glumae lipase have been analyzed in terms of the scooting and hopping models for the action of lipolytic enzymes [Upreti, G.C., & Jain, M.K. (1980) J. Membr. Biol. 55, 113-121]. The results presented in this study are best explained by assuming that glumae lipase leaves the interface after a limited number of catalytic cycles.     

A modified assay of 3':5'-cyclic-AMP phosphodiesterase.

A modification of the assay of cyclic nucleotide phosphodiesterase involving batch use of Dowex 1 anion exchange resin is described which allows for quantitative recovery of adenosine, guanosine, and their metabolites from the resin slurry. The assay described is suitable for use in crude preparations containing purine catabolizing enzymes. A standardized procedure for determining kinetic parameters of cyclic AMP hydrolysis is also discussed. This procedure was used in the partial characterization of the kinetics of cyclic AMP hydrolysis by rat and rabbit heart supernatant fractions.     

Kinetic analysis of the dual phospholipid model for phospholipase A2 action

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.     

Comprehensive kinetic and substrate specificity analysis of an arylsulfatase from Helix pomatia using mass spectrometry

Sulfatases hydrolyze sulfated metabolites to their corresponding alcohols and are present in all domains of life. These enzymes have found major application in metabolic investigation of drugs, doping control analysis and recently in metabolomics. Interest in sulfatases has increased due to a link between metabolic processes involving sulfated metabolites and pathophysiological conditions in humans. Herein, we present the first comprehensive substrate specificity and kinetic analysis of the most commonly used arylsulfatase extracted from the snail Helix pomatia. In the past, this enzyme has been used in the form of a crude mixture of enzymes, however, recently we have purified this sulfatase for a new application in metabolomics-driven discovery of sulfated metabolites. To evaluate the substrate specificity of this promiscuous sulfatase, we have synthesized a series of new sulfated metabolites of diverse structure and employed a mass spectrometric assay for kinetic substrate hydrolysis evaluation. Our analysis of the purified enzyme revealed that the sulfatase has a strong preference for metabolites with a bi- or tricyclic aromatic scaffold and to a lesser extent for monocyclic aromatic phenols. This metabolite library and mass spectrometric method can be applied for the characterization of other sulfatases from humans and gut microbiota to investigate their involvement in disease development.     

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

Lipase-catalyzed hydrolysis of 2-naphtyl esters in biphasic system

The authors measured the rate of hydrolysis of the homologs of 2-naphtyl ester by using a Lewis cell with constant interfacial area to elucidate the kinetic mechanism of the lipase-catalyzed hydrolysis in biphasic system. On the basis of the two-film model, it was found from the analysis of experimental results that the hydrolysis of these substrates proceeds at the interface between the aqueous and organic phases. The interfacial reaction rate could be correlated by Michaelis-Menten mechanism. The values of the rate constant and the Michaelis constant were almost independent of the kinds of 2-naphtyl ester. The values of the interfacial kinetic parameters for 2-naphtyl ester were much greater than those for the hydrolysis in the aqueous phase.     

A direct spectrophotometric γ-glutamyltransferase inhibitor screening assay targeting the hydrolysis-only mode

γ-Glutamyltransferase (GGT, E.C. 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione. Due to its central role in maintaining mammalian glutathione homeostasis, GGT is now believed to be a valuable drug target for a variety of life-threatening diseases, such as cancer. Unfortunately, however, effective tools for screening GGT inhibitors are still lacking. We report here the synthesis and evaluation of an α-phenylthio-containing glutathione peptide mimic that eliminates thiophenol upon GGT-catalyzed hydrolysis of the γ-glutamyl peptide bond. The concurrent, real-time spectrophotometric quantification of the released thiophenol using Ellman’s reagent creates a GGT assay format that is simple, robust, and highly sensitive. The versatility of the assay has been demonstrated by its application to the kinetic characterization of equine kidney GGT, and enzyme inhibition assays. The ability of the glutathione mimic to behave as an excellent donor substrate (exhibiting Michaelis-Menten kinetics with a K_m of 11.3 ± 0.5 μM and a k_cat of 90.1 ± 0.8 nmol mg⁻¹ min⁻¹), coupled to the assay’s ability to study the hydrolysis-only mode of the GGT-catalyzed reaction, make our approach amenable to high-throughput drug screening platforms.     

Insights into the fatty acid chain length specificity of the carboxylesterase PA3859 from Pseudomonas aeruginosa: A combined structural,biochemical and computational study

The open reading frame PA3859 of Pseudomonas aeruginosa encodes an intracellular carboxylesterase belonging to a group of microbial enzymes (EC 3.1.1.1) that catalyze the hydrolysis of aliphatic and aromatic esters with a broad substrate specificity. With few exceptions, for this class of enzymes, belonging to the α/β-hydrolase fold superfamily, very little information is available regarding their biochemical activity and in vivo function. The X-ray crystal structure of recombinant PA3859 has been determined for two crystal forms (space groups P2₁ and P2₁2₁2). The kinetic properties of the enzyme were studied using p-nitrophenyl esters as substrates and data fitted to a surface dilution mixed micelle kinetic model. Enzymatic assays and computational docking simulations, pinpointed the enzyme’s preference for esters of palmitic and/or stearic acids and provided insights into the enzyme–substrate favorable binding modes.     

Purine Nucleoside Phosphorylase Activity in Rat Cerebrospinal Fluid

The sequential hydrolysis of purines is present in rat CSF and generates nucleosides as inosine and guanosine that are usual substrates for purine nucleoside phosphorylase (PNP). PNP catalyzes phosphorolysis of the purine nucleosides and deoxynucleosides releasing purine bases. Here we investigated the presence of PNP in CSF of rats using: i) a specific chromophoric analogue of nucleosides, 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), and ii) an inhibitor of PNP activity, immucillin-H. Additionally, we performed a preliminary kinetic characterization (K(M): Henry-Michaelis-Menten constant; V: maximal velocity) for MESG and inorganic phosphate (Pi). The values of K(M) and V for MESG (n = 3, mean+/-SD) were 142.5+/-29.5 microM and 0.0102+/-0.0006 U mg(-1), respectively. For Pi (n=3, mean+/-SD), the K(M) values and V were 186.8+/-43.7 microM and 0.0104+/-0.0016 U mg(-1), respectively. The results indicated that PNP is present in rat CSF and provided a preliminary kinetic characterization.     

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.     

Kinetic analysis of calcium activation of brain acetylcholinesterase forms.

Calcium activation of acetylcholine hydrolysis by bovine brain acetylcholinesterase (Acetylcholine hydrolase, EC 3.1.1.7) forms has been analyzed in terms of changes in kinetic constants and thermodynamic activation parameters. De-acetylation was determined to be the major rate-influencing step in acetylcholine hydrolysis by both 60 000- and 240 000-dalton forms of the brain enzyme and 10 mM Ca2+ increased the rate constant for this step (k+3) by approximately 30% for both forms. For the smaller acetylcholinesterase form the effects of Ca2+ on de-acetylation was equivalent to its effect on the overall rate constant (k) and occurred without an effect on pK. In the case of the 240 000-dalton species, the overall rate constant was increased by Ca2+ by 33% at pH 8.0 and 81% at pH 7.25 and involved a pK shift of -0.2 pH units. For both enzyme forms the rate constants for acetylation (k+2) were increased by Ca2+. Thermodynamic analysis suggested that Ca2+ activation of the acetylation step was entropically driven. Differences between the two enzymes forms in terms of Ca2+ appear to result from association of low molecular weight species.     

Pancreatic porcine phospholipase A2 catalyzed hydrolysis of phosphatidylcholine in lecithin-bile salt mixed micelles: kinetic studies in a lecithin-sodium cholate system

Pancreatic porcine phospholipase A2 catalyzed hydrolysis of phosphatidylcholine in bile salt lecithin mixed micelles has been studied, utilizing a series of assay mixtures for which the micellar size, weight, and composition had been experimentally determined. Under these conditions the enzymatic hydrolysis is dependent on the phosphatidylcholine-to-sodium cholate molar ratio within the mixed micelle rather than the bulk concentration of the phospholipid in the mixture: at 5 mM phosphatidylcholine, variation of the NPC/NNaCh ratio from 0.2 to 2.0 increases the enzymatic activity from 82 to 933 mumol/min/mg protein. The initial rates are linear throughout the entire series of assay mixtures, the activity vs micellar concentration curves exhibit saturation behavior, and treatment of the data according to the "surface-as-cofactor" theory provides linear double-reciprocal plots which intersect in one point. The assay system should be applicable for detailed kinetic studies of lipolytic enzymes, including mammalian phospholipases which exhibit rather low activities toward lecithin-Triton X-100 mixed micelles. The system should also provide a convenient basis for mechanistic studies involving the use of inhibitory phospholipid substrate analogs.     

Enzyme reactions in a membrane model. 1. A new technique to study enzyme reactions in monolayers

Enzymic hydrolysis by pancreatic phospholipase A (EC 3.1.1.4) of 3-sn-didodecanoyl phosphatidylglycerol was studied under constant surface pressure. A technical device is described which automatically keeps the surface pressure constant and which continuously registers the movement of the surface barrier. A new trough design is proposed which consists of two compartments connected by a narrow surface canal. With this trough the recorded kinetic plots are linear, in contrast to the non-linear kinetic plots obtained with the usual one-compartment trough.     

Isolation, characterization and manipulation of cellulase genes

The complete hydrolysis of cellulose requires a number of different enzymes including endoglucanase, exoglucanase and beta-glucosidase. These enzymes function in concert as part of a 'cellulase'complex called a cellulosome. In order (i) to develop a better understanding of the biochemical nature of the cellulase complex as well as the genetic regulation of its integral components and (ii) to utilize cellulases either as purified enzymes or as part of an engineered organism for a variety of purposes, researchers have, as a first step, used recombinant DNA technology to isolate the genes for these enzymes from a variety of organisms. This review provides some perspective on the current status of the isolation, characterization and manipulation of cellulase genes and specifically discusses (i) strategies for the isolation of endoglucanase, exoglucanase and beta-glucosidase genes; (ii) DNA sequence characterization of the cellulase genes and their accompanying regulatory elements; (iii) the expression of cellulase genes in heterologous host organisms and (iv) some of the proposed uses for isolated cellulase genes.     

Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate

Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0–9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10^− 2 M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000 ± 500 and 2800 ± 60 μmol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240 ± 17 and 432 ± 62 U/mg, respectively) on MGDG.     

Monitoring the timing of ATP hydrolysis with activation of peptide cleavage in Escherichia coli Lon by transient kinetics

Escherichia coli Lon, also known as protease La, is an oligomeric ATP-dependent protease, which functions to degrade damaged and certain short-lived regulatory proteins in the cell. To investigate the kinetic mechanism of E. coli Lon protease, we performed the first pre-steady-state kinetic characterization of the ATPase and peptidase activities of this enzyme. Using rapid quench-flow and fluorescence stopped-flow spectroscopy techniques, we demonstrated that ATP hydrolysis occurs before peptide cleavage, with the former reaction displaying a burst and the latter displaying a lag in product production. The detection of burst kinetics in ATP hydrolysis is indicative of a step after nucleotide hydrolysis being rate-limiting in ATPase turnover. At saturating substrate concentrations, the lag rate constant for peptide cleavage is comparable to the kcat of ATPase, indicating that two hydrolytic processes are coordinated during the first enzyme turnover. The involvement of subunit interaction during enzyme catalysis was detected as positive cooperativity in the binding and hydrolysis of substrates, as well as apparent asymmetry in the ATPase activity in Lon. When our data are taken together, they are consistent with a reaction model in which ATP hydrolysis is used to generate an active enzyme form that hydrolyzes peptide.