Effects of Oxodiperoxovanadate (V) Complexes on the Activity of Green Crab (Scylla serrata) Alkaline Phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in seawater on the activity of the enzyme will result in the loss of the biological function of the enzyme, which will affect the exuviating crab shell and threaten the survival of the animal. In the present paper, the effects of four oxodiperoxovanadate (V) complexes on the activity of green crab alkaline phosphatase have been studied. The results show that these vanadate derivatives can lead to reversible inactivation. The equilibrium constants for binding of inhibitors with the enzyme and/or the enzyme–substrate complexes have been determined. The results show that sodium (2,2'-bipyridine)oxodiperoxovanadate, pV(bipy), and potassium oxodiperoxo-(1,10-phenanthroline)vanadate, pV(phen), are competitive inhibitors, while potassium picolinato-oxodiperoxo-vanadate, pV(pic), and oxalato-oxodiperoxovanadate, pV(ox), are mixed-type inhibitors. These results suggest that pV(bipy) is a considerably more potent competitive inhibitor than pV(phen) and that the competitive inhibition effect of pV(pic) is stronger than that of pV(ox), but the non-competitive inhibition effect of pV(ox) is stronger than that of pV(pic).     

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.Abbreviations ALP alkaline phosphatase - PNPP p-nitrophenyl phosphate - NBS N-bromosuccinimide     

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.     

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.     

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.     

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.     

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.     

Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase by zinc ions: a new type of complexing inhibition

The Tsou method was used to study the kinetic course of inactivation of green crab alkaline phosphatase by zinc ions. The results show that the enzyme was inactivated by a complexing scheme which has not been previously identified. The enzyme first reversibly and quickly binds Zn(2+) and then undergoes a slow reversible course to inactivation and slow conformational change. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is for a saturated reaction being independent of Zn(2+) concentration if the concentration is sufficiently high. The microscopic rate constants of inactivation and the association constant were determined from the measurements.     

Chicken Intestinal Alkaline Phosphatase : I. The kinetics and thermodynamics of reversible inactivation II. Reactivation by zinc ions

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.     

醋酸酐对太平洋白对虾(Penaeus vannamei)β-N-乙酰- D-氨基葡萄糖苷酶的抑制动力学

β-N-乙酰-D-氨基葡萄糖苷酶与南美白对虾的食物消化吸收、蜕壳生长有着密切关系. 海水里存在的有机污染物将影响酶生理功能, 从而进一步影响虾的正常蜕壳,严重将导致对虾的死亡. 醋酸酐是常用的有机溶剂, 故本文应用动力学方法研究醋酸酐对南美白对虾β-N-乙酰-D-氨基葡萄糖苷酶催化pNP-NAG水解时酶活力的变化规律. 表明在醋酸酐浓度低于20.0 mmol/L, 酶的抑制作用是可逆的, 测得醋酸酐对酶抑制的IC50为9.0 mmol/L. 用双倒数作图法测定醋酸酐与游离酶(E)和酶-底物络合物(ES)的结合平衡常数, 结果显示醋酸酐是酶的非竞争性抑制剂. 用底物反应动力学方法观测在不同底物浓度下酶在0.0、3.0、6.0、9.0、12.0 mmol/L的醋酸酐溶液中的失活过程,分别测定了酶的微观失活速度常数k+0及复活速度常数k-0, 结果表明醋酸酐对酶的影响是快速结合再缓慢失活的过程. 比较微观失活速度常数k+0及复活速度常数k-0, 结果暗示在高浓度的醋酸酐溶液中, 酶将完全失活.     

Kinetics of complexing activation by the magnesium ion on green crab (Scylla serrata) alkaline phosphatase.

As with mammalian enzymes, green crab (Scylla serrata) alkaline phosphatase can be activated by Mg2+ through a time-dependent course. The activation is mainly a Vmax effect. Tsou's method was used to study the kinetic course of activation. The results show that the enzyme was activated by a complexing scheme that had not been previously identified: the enzyme first reversibly and quickly binds Mg2+ and then undergoes a slow reversible course to activation, with a relatively high activation energy (78 +/- 4 kJ/mol) and a slow conformational change. The activation reaction is a single molecule reaction, and the apparent activation rate constant is independent of Mg2+ concentration if the concentration is sufficiently high. The microscopic rate constants of activation and the association constant were determined from the measurements. The proposed scheme may also be applied to the Mg2+ activation mechanism for mammalian enzyme, to explain why the activation rate is time-dependent and not diffusion controlled. Substrate binding was also shown to affect the activation rate constant.     

The inactivation of thymidylate synthase by periodate

Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of IO4- at 0 degree C. Nearly complete inhibition resulted when the IO4- concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO4- was in excess. The substrate dUMP, the product dTMP, and inorganic phosphate all protected the enzyme from inactivation by IO4-, with the order of effectiveness: dUMP greater than dTMP greater than phosphate. Deoxyuridine, which is not a substrate, did not protect the enzyme. Titrations with dithiobis(2-nitrobenzoate) (DTNB) showed that approximately 1.5 titratable SH groups were lost when thymidylate synthase was completely inhibited by IO4-. Essentially no reactivation occurred when periodate-inhibited enzyme was dialyzed against buffered 2-mercaptoethanol (ME) or dithiothreitol (DTT). Enzyme that had been treated with p-hydroxymercuribenzoate, DTNB, or methylmethanethiosulfonate prior to treatment with periodate could be completely reactivated with ME or DTT.     

Inhibitory kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei) by zinc ion

Prawn (Litopenaeus vannamei) beta-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) is involved in the digestion and molting processes. Zinc is one of the most important metals often found in the pollutant. In this article, the effects of Zn(2+) on prawn NAGase activity for the hydrolysis of pNP-NAG have been investigated. The results showed that Zn(2+) could reversibly and noncompetitively inhibit the enzyme activity at appropriate concentrations and its IC(50) value was estimated to be 6.00 +/- 0.25 mM. The inhibition model was set up, and the inhibition kinetics of the enzyme by Zn(2+) has been studied using the kinetic method of the substrate reaction. The inhibition constant was determined to be 11.96 mM and the microscopic rate constants were also determined for inactivation and reactivation. The rate constant of the inactivation (k(+0)) is much larger than that of the reactivation (k(-0)). Therefore, when the Zn(2+) concentration is sufficiently large, the enzyme is completely inactivated. On increasing the concentration of Zn(2+), the fluorescence emission peak and the UV absorbance peak are not position shifted, but the intensity decreased, indicating that the conformation of Zn(2+)-bound inactive NAGase is stable and different from that of native NAGase. We presumed that Zn(2+) made changes in the activity and conformation of prawn NAGase by binding with the histidine or cysteine residues of the enzyme.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence,reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 °C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

Molecular asymmetry in alkaline phosphatase of Escherichia coli

Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes. In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively. Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme. Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated. The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties. Structural implications of these results have been discussed.     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by sodium (2,2'-bipyridine) oxodiperoxovanadate

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by sodium (2, 2-bipyridine) oxodiperoxovanadate, pV(bipy), has been studied. The time course of the hydrolysis of p-nitrophenyl-phosphate catalyzed by the enzyme in the presence of different pV(bipy) concentrations showed that at each pV(bipy) concentration, the rate decreased with increasing time until a straight line was approached, the straight line slopes are the same for all concentrations. The results suggest that the inhibition of the enzyme by pV(bipy) is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction of inhibitor with the enzyme.     

The role of Zn(II) in calf intestinal alkaline phosphatase studied by the influence of chelating agents and chemical modification of histidine residues.

Alkaline phosphatase from calf intestine (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is reversibly inhibited at pH 8.0 by incubation with chelating agents. Complete reactivation may be achieved by stoichiometric addition of Zn2+. Atomic absorption spectrometry was used to demonstrate the linear correlation between Zn2+ content and degree of reactivation. The reversibly inhibited enzyme contained 1 Zn2+ per subunit whereas 2 Zn2+ were found in both the reactivated and the native enzyme. At more alkaline pH-values, inactivation by chelating agents becomes irreversible; under such conditions the inactivated alkaline phosphatase still contains 1 Zn2+ per subunit. The conformational changes resulting from the loss of Zn2+ and leading to irreversible inactivation were investigated by optical rotatory dispersion, immunological techniques, and ultraviolet and fluorescence spectroscopy. Azocoupling of the alkaline phosphatase with diazonium-1-H-tetrazole and Zn2+ content measurement of azocoupled enzyme probes indicated that 2 histidine residues per subunit are involved in binding of the catalytically important Zn2+.     

Inhibition of Escherichia coli glutamine synthetase by alpha- and gamma-substituted phosphinothricins

We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.     

A study of dithiothreitol inactivation of the enzyme rhodanese.

When air oxidized, partially inactivated rhodanese (EC 2.8.1.1) is treated with dithiothreitol (DTT) to regenerate the reduced essential sulfhydryl group there is an initial reactivation followed by an anomalous slower inactivation. Fully active enzyme shows only inactivation. The inactivated enzyme may be completely reactivated on long incubation with the substrate thiosulfate ion. None of the normal potentialities of DTT appear to be responsible for the inactivation. The results are interpreted in terms of disulfide formation between DTT and an essential enzymic sulfhydryl group with the resulting complex being stabilized by secondary interactions which are particularly favorable due to similarities between DTT and lipoic acid--a normal sulfur acceptor substrate.     

Kinetic and mechanism of interaction of L-cysteine with alkaline phosphatase from calf intestines

The effect of L-cysteine on activity of hydrophobic forms of calf intestine alkaline phosphatase was investigated. Apparent inhibition constants for mixed type inhibition have been determined. The kinetic results allow supposing that the mechanism of equilibrium establishment between the inhibitor and enzyme involves the initial rapid formation of intermediate complex and a subsequent slower step leading to its stabilization in the substrate binding site. The microscopic rate constants for slow step of interaction of L-cysteine with alkaline phosphatase have been calculated. Effect of pH on apparent inhibition constants and kinetic parameters for enzymatic reaction in the presence of L-cysteine was analysed.