Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.     

Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction

A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldoxime methiodide (2-PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Irreversible kinetics of β-N-acetyl-d-glucosaminidase from prawn (Penaeus vannamei) inactivated by mercuric ion

Cysteine residues in prawn (Penaeus vannamei) β-N-acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) have been modified by p-chloromercuribenzoate (PCMB). The results show that sulfhydryl group is essential for the activity of the enzyme. Inactivation kinetics of the enzyme by mercuric chloride (HgCl₂) has been studied using the kinetic method of the substrate reaction during inactivation of enzyme previously described by Tsou. The kinetic results show that the inactivation of the enzyme is an irreversible reaction. The microscopic rate constants for the reaction of Hg²⁺ with free enzyme and with the enzyme-substrate complex are determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by Hg²⁺. The above results suggest that the cysteine residue is essential for activity.     

New insights into the catalytic mechanism of human glycine N‐acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.     

Properties of halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. True Michaelis constants, reaction mechanisms and molecular weights

True values of Michaelis constants of the NADP(+)-specific isocitrate dehydrogenase from Halobacterium salinarium were not very different from those of the apparent constants reported by Aitken et al. (1970). The true constants were affected by salt in a similar manner to that of the apparent constants obtained with NADP(+) at fixed concentrations of 1.0-0.2mm and threo-d(s)-(+)-isocitrate at fixed concentrations of 2.0-0.125mm. The response of apparent V(max.) to salt concentration was highly dependent on fixed substrate concentration in solutions of sodium chloride but much less so in solutions of potassium chloride. At several levels the results emphasize the difficulty of generalizing about the salt relations of a halophil enzyme without adequate attention to substrate concentration. The enzyme has at least two different reaction mechanisms depending on salt concentration. In its ;physiological' form (i.e. in 1.0m-potassium chloride), and also in 1.0m-sodium chloride, the reaction mechanism is ordered with NADP(+) the first substrate added and NADPH the last product released. In 0.25m-sodium chloride, however, the mechanism is different and is probably non-sequential. In 4.0m-sodium chloride with low concentrations of either fixed substrate, there was evidence of a co-operative action of the variable substrate. The evidence suggests that salt participates in the reaction mechanism in two ways: one is the reversible addition to the enzyme in a manner analogous to that of a substrate; the other is dead-end complex-formation. The relative contributions of these two types of reaction determine whether salt activates or inhibits the enzyme. In addition, the inhibition caused by high concentrations of sodium chloride is more complex than the corresponding inhibition by potassium chloride. Gel-filtration experiments indicated that at very low salt concentrations the enzyme has an apparent molecular weight of about 70800. In ;physiological' concentrations of potassium chloride the enzyme appears to be a dimer (mol.wt. 122000-135000) and, in 1.0-4.0m-sodium chloride, it behaves as a trimer or tetramer (mol.wt. 224000-251000). A preliminary method of purifying the enzyme is described.     

Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase.

Mitochondrial NADH dehydrogenase has been purified to homogeneity by resolution of Complex I from beef heart mitochondria with the chaotrope NaClO₄ and precipitation of the enzyme with ammonium sulfate. The enzyme is water-soluble, has a molecular weight of 69,000 ± 1000 as determined by gel filtration on Sephadex G-100 and agarose 1.5 M. It is an iron-sulfur flavoprotein, with the ratio of flavin (FMN) to nonheme iron to labile sulfide being 1:5–6:5–6. The FMN content suggests a minimum molecular weight of 74,000 ± 3000 for the enzyme. NADH dehydrogenase is composed of three subunits with apparent M_r values, as determined by acrylamide gel electrophoresis as well as by gel filtration on agarose 5 M both in the presence of sodium dodecyl sulfate, of about 51,000, 24,000, and 9–10,000. Coomassie blue stain intensities of the subunits on acrylamide gels suggest that they are present in NADH dehydrogenase in equimolar amounts. However, summation of the apparent M_r values of the dodecyl sulfate-treated subunits appears to overestimate the molecular weight of the native enzyme. The amino acid compositions of NADH dehydrogenase and of each of the isolated and purified subunits have been determined. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH by quinones, ferric compounds, and NAD (3-acetylpyridine adenine dinucleotide was used). All the activities of NADH dehydrogenase are greatly stimulated by addition of guanidine (up to 150 mm), alkylguanidines, arginine, and arginine methyl ester to the assay medium. Phosphoarginine had no effect. These results pointed to the importance of the positively charged guanido group, which appears to interact with and neutralize the negative charges on NAD(P)H and thereby allow for better enzyme-substrate interaction. In the absence of guanidine, NADPH is essentially unoxidized by the enzyme at pH values above 6.0. However, both NADPH dehydrogenase and NADPH → NAD transhydrogenase activities increase dramatically as the assay pH is lowered below pH = 6. Since the pK of the 2′-phosphate of NADPH is 6.1, it appears that the above pH effect is related to protonation of the 2′-phosphate, thus rendering NADPH a closer electronic analog of NADH, which is the primary substrate of the enzyme.     

Kinetics of modification of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid)

The kinetic theory of the substrate reaction during modification of enzyme activity previously described by Tsou [Tsou (1988),Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436] has been applied to a study of the kinetics of the course of inactivation of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid) (DTNB). The results show that the inactivation of this enzyme by DTNB is a conformation-change-type inhibition which involves a conformational change of the enzyme before inactivation. The microscopic rate constants were determined for the reaction of the inactivator with the enzyme. The presence of the substrate provides marked protection of this enzyme against inactivation by DTNB. The modification reaction of the enzyme using DTNB was shown to follow a triphasic course by following the absorption at 412 nm. Among these reactive thiol groups, the fast-reaction thiol group is essential for the enzyme activity. The results suggest that the essential thiol group is situated at the succinate-binding site of the mitochondrial succinate-ubiquinone reductase.     

A rapid and sensitive fluorescence assay for angiotensin-converting enzyme.

A new, highly sensitive, specific assay for dopamine-β-hydroxylase (DBH) activity in human serum is described. Tyramine is used as a substrate; the product of the enzymatic hydroxylation, octopamine, is converted by reacting with 1-dimethylaminonaphthalene-5-sulfonyl-chloride (Dns-Cl) to a fluorescent product, which is extracted from the reaction mixture and purified from the extract by thin-layer chromatography (tlc). The fluorescence of the dansylated octopamine is measured in situ on the tlc plate using a chromatogram-spectrofluorometer. This one-step enzyme reaction can be performed at optimum pH and substrate concentration. As little as 8 ng of octopamine can be determined accurately; the response is linear up to more than 400 ng of octopamine. A comparison with the radioenzymatic assay (Weinshilboum, R., and Axelrod, J. (1971) Circ. Res.28, 307–315) shows an approximately twofold increase in the enzymatic activity measured. Kinetic studies of human sera with high and low DBH activity gave a K_m value of 3.1 × 10^?3m. The method is successfully being used for the functional characterization of the enzyme and genetic studies (Herschel, M., in preparation).     

Irreversibly inhibitory kinetics of 3,5-dihydroxyphenyl decanoate on mushroom (Agaricus bisporus) tyrosinase

3,5-Dihydroxyphenyl decanoate (DPD) is found to inhibit the diphenolase activity of tyrosinase from mushroom (Agaricus bisporus). The effects of DPD on the diphenolase activity of mushroom tyrosinase have been studied. The results show that the enzyme activity decreases very slowly with an increase in DPD concentrations at lower concentrations of DPD (between 5 and 60 microM). But at higher concentrations of DPD, DPD can strongly inhibit the diphenolase activity of the enzyme and the inhibition is irreversible. The IC50 value was estimated to be 96.5 microM. The inhibition mechanism of DPD has been investigated and the results show that DPD can bind to the free enzyme molecule and enzyme-substrate complex and lose the enzyme activity completely. The inhibition kinetics has been studied in detail by using the kinetic method of the substrate reaction described by Tsou. The microscopic rate constants of the enzyme inhibited by DPD at higher concentrations have been determined.     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.     

A homogeneous isoenzyme of human liver acid phosphatase.

An isoenzyme of human liver acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) has been purified 4560-fold to homogeneity. The purification procedure includes ammonium sulfate fractionation, acid treatment, ion exchange chromatography on O-(carboxymethyl)-cellulose and DEAE-cellulose, Sephacryl S-200 chromatography, and affinity chromatography on Concanavalin A-Sepharose 4B. The homogeneous enzyme is a glycoprotein having 4% carbohydrate by weight in the form of mannose and glucosamine. In polyacrylamide gel electrophoresis under varied conditions of pH and cross-linking, the purified enzyme displays a single protein band coincident with activity. The native enzyme has a molecular weight of 93,000 as determined by gel elution chromatography and consists of two equivalent polypeptide chains. The subunit weight is 50,000–52,000 by sodium dodecyl sulfate gel electrophoresis. l-(+)-Tartrate is a strong competitive inhibitor of the enzyme; K_i is 4.3 × 10^?7m at pH 4.8 in 50 mm sodium acetate/100 mm sodium chloride. K_i values for a number of other inhibitors are given. Although it catalyzes the hydrolysis of a variety of phosphomonoesters, this isoenzyme of human liver acid phosphatase does not hydrolyze adenosine 5′-diphosphate, adenosine 5′-triphosphate, pyrophosphate, or choline phosphate at a detectable rate. The values of V differ with different alcohol or phenol leaving groups. The pH dependence of K_m and V values for the hydrolysis of p-nitrophenyl phosphate have been determined together with the pH dependence of K_i for l-(+)-tartrate. The pH dependence of both K_m and V indicate the effect of substrate ionization (pK ~ 5.2) and the involvement of a group in the EScomplex having a pKa value of approximately 6–7 which is ascribed either to a phosphoryl-enzyme intermediate or to the ionization of substrate in the ES-complex. An irreversible modification of the enzyme and a rapid loss of enzymic activity occurs upon treatment of the enzyme with Woodward's reagent K. The enzyme is protected against inactivation by the presence of competitive inhibitors. These and other data suggest that at least one carboxylic acid group plays an important role in catalysis.     

Enzyme kinetics: partial and complete uncompetitive inhibition

A graphical method for analysing enzyme data to obtain kinetic parameters, to identify the types of inhibition and the enzyme mechanisms is described. The method consists of plotting experimental data as v/(V(0)-v) versus 1/(I) at different substrate concentrations. I is the inhibitor concentration; V(0) and v are the initial rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate and in the absence and presence of inhibitor respectively. Complete inhibition gives straight lines that pass through the origin while partial inhibition gives straight lines that converge on the 1/I-axis at a point away from the origin. With uncompetitive inhibition the slopes of the lines decrease with increasing substrate concentration. The kinetic parameters K(m), K'(i) and beta (degree of partiality) can best be determined from respective secondary plots of slope and intercept versus reciprocal of substrate concentration.     

Uricase from soybean root nodules: Purification,properties, and comparison with the enzyme from cowpea

A 45-fold purification of uricase (urate:O₂ oxidoreductase, EC 1.7.3.3) from soybean root nodules by ammonium sulfate fractionation, gel filtration, and affinity chromatography is described. Electrophoresis on nondenaturing gels using an activity stain or on sodium dodecyl sulfate (SDS) gels demonstrated that the enzyme obtained was nearly homogeneous. The subunit molecular weight of uricase estimated from SDS gels was 32,000 ± 3000. Gel-filtration studies indicated that the native enzyme is a monomer at pH 7.5 which associates to form a dimer at pH 8.8. Enzyme activity was stabilized by the addition of dithiothreitol. The pH dependence of the enzyme showed an optimum of 9.5. Initial rate kinetics showed K_m values of 10 and 31 μm for uric acid and oxygen, respectively, with an intersecting pattern of substrate dependence. Uricase activity was inhibited strongly by xanthine, which was competitive with respect to uric acid (K_i = 10 μm). No significant inhibition was observed in the presence of a variety of amino acids, ammonium, adenine, or allopurinol, in contrast with results reported for the cowpea enzyme. Gel-filtration chromatography and SDS-gel electrophoresis of uricase purified by the same method from cowpea nodules indicated that the native enzyme exists as a monomer of M_r 50,000 at pH 7.5.     

Utilization of Selenocysteine by a Cysteinyl-tRNA Synthetase from Phaseolus aureus

An l-cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus has been purified approximately 200-fold. The enzyme uses selenocysteine as substrate in the ATP-PPi exchange assay; other cysteine analogs were inactive. The molecular weight as determined by Sephadex G-200 column chromatography is about 61,000; sodium dodecyl sulfate and 8 m urea acrylamide gel electrophoresis indicate that the enzyme is a dimer consisting of two identical monomers of molecular weight 30,000. A method for the preparation of selenocysteine from selenocystine is described.     

Mechanistic and kinetic studies of inhibition of enzymes

A graphical method for analyzing enzyme data to obtain kinetic parameters, and to identify the types of inhibition and the enzyme mechanisms, is described. The method consists of plotting experimental data as nu/(V0 - nu) vs 1/(I) at different substrate concentrations. I is the inhibitor concentration; V0 and nu are the rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate, and in the absence and presence of inhibitor, respectively. Complete inhibition gives straight lines that go through the origin; partial inhibition gives straight lines that converge on the 1-I axis, at a point away from the origin. For competitive inhibition, the slopes of the lines increase with increasing-substrate concentration; with noncompetitive inhibition, the slopes are independent of substrate concentration; with uncompetitive inhibition, the slopes of the lines decrease with increasing substrate concentrations. The kinetic parameters, Km, Ki, Ki', and beta (degree of partiality) can best be determined from respective secondary plots of slope and intercept vs substrate concentration, for competitive and noncompetitive inhibition mechanism or slope and intercept vs reciprocal substrate concentration for uncompetitive inhibition mechanism. Functional consequencs of these analyses are represented in terms of specific enzyme-inhibitor systems.     

Type II NADH dehydrogenase of the respiratory chain of Plasmodium falciparum and its inhibitors

Plasmodium falciparum NDH2 (pfNDH2) is a non-proton pumping, rotenone-insensitive alternative enzyme to the multi-subunit NADH:ubiquinone oxidoreductases (Complex I) of many other eukaryotes. Recombinantly expressed pfNDH2 prefers coenzyme CoQ₀ as an acceptor substrate, and can also use the artificial electron acceptors, menadione and dichlorophenol–indophenol (DCIP). Previously characterized NDH2 inhibitors, dibenziodolium chloride (DPI), diphenyliodonium chloride (IDP), and 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) do not inhibit pfNDH2 activity. Here, we provide evidence that HDQ likely targets another P. falciparum mitochondrial enzyme, dihydroorotate dehydrogenase (pfDHOD), which is essential for de novo pyrimidine biosynthesis.     

Purification and properties of a homogeneous aryl sulfatase A from rabbit liver.

Aryl sulfatase A (aryl sulfate sulfohydrolase EC 3.1.6.1) has been purified > 10,000-fold from rabbit liver; by disc gel electrophoresis the enzyme appears homogeneous. Various properties of the enzyme have been determined and comparisons are made with other aryl sulfatases. Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is made up of monomers of molecular weight ～ 70,000. At pH 7.4 the enzyme exists as a dimer whereas a tetrameric form predominates at pH 4.8.The enzyme exhibits the anomalous kinetics often observed with aryl sulfatase A from mammalian tissues (the enzyme is modified to an inactive form while degrading substrate and the inactive form can be reactivated by sulfate ion). The enzyme activity has been studied under a variety of reaction conditions. Two pH optima are observed and neither enzyme concentration or changes in ionic strength appear to have an effect on the relative magnitudes of the optima. Aryl sulfatase A is competitively inhibited by potassium sulfate, potassium phosphate, and sodium sulfite (K_i = 2.9 × 10^?3 M, 3.4 × 10^?5 M, and 1.1 × 10^?6 M, respectively). Kinetic constants for some substituted phenyl sulfate esters have been determined. The variation in V is not consistent with a reaction mechanism involving a rate-limiting breakdown of a common intermediate.The inactive (modified) form of the enzyme has been isolated from reaction mixtures containing aryl sulfatase A and substrate. A procedure is presented for determining the relative amount of modified and native enzyme in these preparations. In the presence of substrate, sulfate displaces the equilibrium between native and modified enzyme in favor of native enzyme. In the absence of substrate neither sulfate or phosphate have an effect on the equilibrium. A study is made of the temperature dependence of the process in which the modified enzyme is converted back to native enzyme. The relatively small entropy of activation for the conversion of the modified to the native form (ΔS3 = ?8 cal/mole deg) does not seem to be consistent with a major modification of protein conformation.     

Purification and some properties of Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase

Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19) was purified by the technique of starch adsorption and DEAE-cellulose column chromatography, and then crystallized from an ammonium sulfate solution containing mM calcium chloride. The crystals of the enzyme were rod-shaped and showed a single band by disc-gel electrophoresis. The purified enzyme was dissociated into two subunits by sodium dodecyl sulfate-disc electrophoresis. The subunits had no enzyme activity. Details of each purification step and some properties of the enzyme are described in this paper.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.