Effect of vanadate on phosphoryl transfer enzymes involved in glucose metabolism

Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate. Phosph$\text{o}$ glucomutase and phosphoglycerate mutase are inhibited at the μM range. 2,3-Bisphosphoglycerate phosphatase is completely inhibited by 0.5 mM vanadate. 2,3-Bisphosphoglycerate synthase, hexokinase, phosphoglycerate kinase and fructose-1,6-P₂ phosphatase are partially inhibited by mM vanadate. Phosphofructokinase and pyruvate kinase are not affected. The glycolytic enzymes which mechanism does not involves phosphoryl transfer step are not affected by vanadate.     

Efficient,crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer

The observation that one enzyme can accelerate several chemically distinct reactions was at one time surprising because the enormous efficiency of catalysis was often seen as inextricably linked to specialization for one reaction. Originally underreported, and considered a quirk rather than a fundamental property, enzyme promiscuity is now understood to be important as a springboard for adaptive evolution. Owing to the large number of promiscuous enzymes that have been identified over the last decade, and the increased appreciation for promiscuity's evolutionary importance, the focus of research has shifted to developing a better understanding of the mechanistic basis for promiscuity and the origins of tolerant or restrictive specificity. We review the evidence for widespread crosswise promiscuity amongst enzymes that catalyze phosphoryl transfer, including several members of the alkaline phosphatase superfamily, where large rate accelerations between 10⁶ and 10¹⁷ are observed for both native and multiple promiscuous reactions. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

Aqueous interactions of vanadate and peroxovanadate with dithiothreitol. Implications for the use of this redox buffer in biochemical investigations

 Dithiothreitol (CH₂SHCHOHCHOHCH₂SH), under neutral conditions in aqueous medium, reacts readily and reversibly with vanadate to form longlived complexes. The ligand, vanadium and proton stoichiometries were established from concentration and pH studies. The two predominant products each contained two vanadium(V) nuclei and one dithiothreitol and carried an overall doubly negative charge. The equilibrium shifted toward a triply negative charge with increase in pH through the pK _a range of the products. The ⁵¹V NMR spectra clearly showed two resonances for each product (–352 and –362 ppm for one and –399 and –526 ppm for the other), thus establishing there are chemical differences in the coordination about each vanadium. A coordination scheme was proposed for each product. The common motif proposed was the presence of a cyclic [VO]₂ core as the source of a strong stabilizing interaction leading to the very favourable formation constants (overall about 10⁷ at pH 7). The coordination shell about the individual vanadiums each contained one sulfur in the one product and one sulfur about one vanadium and only oxygen about the other vanadium in the second product. Under neutral conditions the reduction of V(V) to V(IV) requires in the order of 90 min. However, if hydrogen peroxide, in greater than a 2 : 1 molar ratio over dithiothreitol, is included in the reaction medium, all the dithiothreitol is rapidly oxidized, and peroxovanadium(V) complexes are observed. Addition of excess dithiothreitol regenerates the dithiothreitol/vanadate complexes. Received: 2 May 1997 / Accepted: 2 July 1997     

The effect of vanadate upon the expression of phenylalanine hydroxylase in streptozotoxin-diabetic rat liver

Induction of diabetes in rats is associated with a significant elevation in the phenylalanine hydroxylating capacity of the liver. This phenomenon reflects an increase in the abundance of both phenylalanine hydroxylase protein and phenylalanine hydroxylase-specific mRNA. These changes can be abolished by insulin-dependent control of diabetes. We show here that the control of diabetes of oral administration of sodium orthovanadate will also nullify the diabetes-related alterations in phenylalanine hydroxylase expression. In addition, diabetes-induced changes in the extent of phosphorylation of phenylalanine hydroxylase are reversed by either or vanadate treatment in vivo. These treatments also abolished the diabetes-related, approx. 30-fold, decrease in glucagon sensitivity of phenylalanine hydroxylation in isolated liver cells.     

The effects of vanadate on calcium transport in dialyzed squid axons Sidedness of vanadate-cation interactions

(1) Vanadate (VO₃^?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Ca_o. External Ca lead to a release of the inhibitory effect. ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{mM}$). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K⁺ increases the affinity of the intracellular vanadate binding site. External K⁺ has no effect on the inhibition. (4) Vanadate has no effect on the Na_o-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.     

The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase

The second order rate constant (k(cat)/K(m)) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping (14)CO(2) released during the reaction, is 2 x 10(-4)M(-1)s(-1). This very low activity may be compared with a value of 3 x 10(7)M(-1)s(-1) for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (K(i)=7 x 10(-4)M), imply an effective concentration of 1.1 x 10(9)M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 x 10(11)-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.     

Influence of vanadate on calcium fluxes and net movement of calcium in intact squid axons

Intracellular vanadate at a concentration of 100 μM inhibits the uncoupled efflux of Ca²⁺ from intact axons but has little effect on the exchange fluxes and on the Ca²⁺-dependent Na⁺ efflux. External vanadate has no effect on the Ca²⁺ efflux. In addition and most importantly intracellular vanadate inhibits the Ca²⁺ efflux in the presence of external Na⁺ and Ca²⁺ suggesting that the uncoupled efflux is operative under physiological conditions. Measurements of the net movements of Ca²⁺ under near physiological conditions have confirmed this conclusion.     

Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate

Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca²⁺ solution (pCa> 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca2²⁺ (pCa 4.5 prior to incubation with vanadate. Transfer back to a high Ca²⁺ solution elicited no additional force (83.2% ± 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.     

Computational study of a transition state analog of phosphoryl transfer in the Ras–RasGAP complex: AlF x versus MgF 3 –

The structures of the complexes between Ras•GDP bound to RasGAP in the presence of three probable γ-phosphate analogs (AlF₃, AlF₄⁻ and MgF₃⁻) for the transition state (TS) of the hydrolysis of guanosine triphosphate (GTP) by the Ras-RasGAP enzymes have been modeled by quantum mechanical—molecular mechanical (QM/MM) calculations. These simulations contribute to the dispute on the nature of the TS in the hydrolysis reaction, since medium resolution X-ray crystallography cannot discern among stereochemically similar isoelectronic species (e.g., AlF₃ or MgF₃⁻). The optimized geometry for each structure has been found starting from experimental coordinates of one of them (PDBID: 1WQ1). Direct comparison of the experimental and computed geometry configurations in the immediate vicinity of the active site suggests that MgF₃⁻ is the most likely candidate for the phosphate analog in the experimental structure.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Effect of long-term treatment with vanadate in drinking water on KK mice with genetic non-insulin-dependent diabetes mellitus

The glucose-lowering effect of vanadate, ammonium metavanadate (AMV), on diabetic KK mice was examined. Five-week-old male KK mice were administrated with a solution of AMV via drinking water at concentrations of vanadium (V) with 0.1, 1.0, 10 and 100 μg/mL for a period of 10 wk, respectively. Body weight, consumption of food and water, and blood glucose levels was measured every week for 10 wk. The results showed that food consumption and body weight in the experimental groups were similar to those in the control group. A statistically significant decrease of drinking water consumption and blood glucose levels in the group treated with 100 μg V/mL was observed. The glucose tolerance in the vanadate-treated mice with 10 and 100 μg V/mL was remarkably improved compared with the control group. Biochemical analyses at the end of experiments demonstrated that a distinct tendency for the glucose and hemoglobin A1c (HbA1c) levels to decrease with vanadate treatment in the blood was also observed. The glutamic pyruvic transaminase, glutamic oxaloacetate transaminase, blood urea nitrogen, triglyceride, high-density lipoprotein, and total cholesterol levels in plasma were lower in the higher vanadium groups than those in the control group. These results indicate that vanadium effectively produced the glucose-lowering effect at a higher dose than that at a low dose of vanadium in drinking water, without any overt signs of toxicity.     

Separation of bisdioxopiperazine- and vanadate resistance in topoisomerase II

Bisdioxopiperazines are inhibitors of topoisomerase II trapping this protein as a closed clamp on DNA with concomitant inhibition of its ATPase activity. Here, we analyse the effects of N-terminal mutations identified in bisdioxopiperazine-resistant cells on ATP hydrolysis by this enzyme. We present data consistent with bisdioxopiperazine resistance arising by two different mechanisms; one involving reduced stability of the N-terminal clamp (the N-gate) and one involving reduced affinity for bisdioxopiperazines. Vanadate is a general inhibitor of type P ATPases and has recently been demonstrated to lock topoisomerase II as a salt-stable closed clamp on DNA analogous to the bisdioxopiperazines. We show that a R162K mutation in human topoisomerase II alpha renders this enzyme highly resistant towards vanadate while having little effect on bisdioxopiperazine sensitivity. The implications of these findings for the mechanism of action of bisdioxopiperazines versus vanadate with topoisomerase II are discussed.     

Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.     

Oxidative stress in toadfish (Halobactrachus didactylus) cardiac muscle: Acute exposure to vanadate oligomers

Vanadate solutions as ‘metavanadate’ (containing ortho and metavanadate species) and ‘decavanadate’ (containing manly decameric species) (5 mM; 1 mg/kg) were injected intraperitoneously in Halobatrachus didactylus (toadfish), in order to evaluate the contribution of decameric vanadate species to vanadium (V) intoxication on the cardiac tissue. Following short-term exposure (1 and 7 days), different changes on antioxidant enzyme activities—superoxide dismutase (SOD), catalase (CAT), selenium-glutathione peroxidase (Se-GPx), total glutathione peroxidase (GPx), lipid peroxidation and subcellular vanadium distribution were observed in mitochondrial and cytosolic fractions of heart ventricle toadfish. After 1 day of vanadium intoxication, SOD, CAT and Se-GPx activities were decreased up to 25%, by both vanadate solutions, except mitochondrial CAT activity that increased (+23%) upon decavanadate administration. After 7 days of exposure, decavanadate versus metavanadate solutions promoted different effects mainly on cytosolic CAT activity (−56% versus −5%), mitochondrial CAT activity (−10% versus +10%) and total GPx activity (+1% versus −35%), whereas lipid peroxidation products were significantly increased (+82%) upon 500 μM decavanadate intoxication. Accumulation of vanadium in total (0.137±0.011 μg/g) and mitochondrial (0.022±0.001 μg/g) fractions was observed upon 7 days of metavanadate exposure, whereas for decavanadate, the concentration of vanadium increased in cytosolic (0.020±0.005 μg/g) and mitochondrial (0.021±0.009 μg/g) fractions. It is concluded that decameric vanadate species are responsible for a strong increase on lipid peroxidation and a decrease in cytosolic catalase activity thus contributing to oxidative stress responses upon vanadate intoxication, in the toadfish heart.     

Studies on propylamine transfer activity in anti-AdoDATO antibodies

Summary Based on a structural similarity to the transition state of a propylamine transfer reaction involved in polyamine biosynthesis, Sadenosyl-(5)-1,8-diamino-3-thiooctane (AdoDATO), the most potent inhibitor of spermidine synthases, was used as a hapten for mice immunization. From immunized mice sera, the IgG fractions were purified by means of affinity (protein A/G) chromatography. Sera and purified polyclonal antibodies from several mice were found to exert spermidine synthase-like activity. Moreover, by means means of hybridoma technology, 19 anti-AdoDATO hybridoma clones have been screened for propylamine transfer activity and at least 6 were found to produce catalytic antibodies. These findings indicate the presence in the sera of active spermidine synthase-like catalytic antibodies. The reported results for the first time evidence the feasibility of preparation of N-alkylating antibodies, widening the biotechnological perspectives of antibodies as biocatalysts.Abbreviations AdoDATO S-adenosyl-(5)-1,8-diaminothiooctane - TSA Transition state analog - decAdoMet S-adenosyl-(5)-3-methylthiopropylamine (decarboxylated adenosylmethionine) - KLH keyhole limpet hemocyanin - NMR nuclear magnetic resonance - BSA bovine serum albumine - WSC 1-ethyl-3-(dimethylaminopropyl)carbodiimide (water-soluble carbodiimide) - PBS phosphate buffer saline - OPD orthodiphenylenediamine - TCA trichloroacetic acid - SDS-PAGE sodium duodecylsulphate-polyacrylamide gel electrophoresis - SN2 bimolecular nucleophilic substitution; abzyme catalytic antibody - IgG immunoglobulin G     

pH modulation of transient state kinetics of enzymes. I. Simple theoretical models

The effect of proton concentration on pre-steady-state kinetics has been investigated theoretically for enzyme reactions involving the breaking of one substrate into two products. Even for the simple double-intermediate mechanism the approach to the steady state may exhibit a rather complex kinetics, which is pH-dependent. This process may even exhibit damped oscillations. A change of pH may completely change this transient kinetics and even suppresses the oscillatory regime. A simple method is presented which allows estimation of the values of the rate and ionization constants. This procedure allows one to distinguish the simple double-intermediate mechanism from a more complex process where the 'fast' binding of the substrate induces a 'slow' conformation change of the enzyme.     

陆生植物体内酶系统对UV-B辐射增强的响应

臭氧层减薄导致地表中波紫外线UV-B(280～320 nm)辐射的增强,UV-B辐射能量远高于可见光,且能被植物体内蛋白质和核酸等生物大分子吸收.酶是植物体内起催化作用的一类蛋白质,酶的数量和活性对UV-B辐射增强有强烈的响应.本文将近年来增强UV-B辐射对植物体内酶影响的研究工作进行了综述.主要包括抗氧化酶、核酮糖-1,5-二磷酸羧化酶、硝酸还原酶和谷氨酰胺合成酶.并就今后该方面的研究提出建议.