Interaction of Divalent Metal Ions with Zn2+-Glycerophosphocholine Cholinephosphodiesterase from Ox Brain

The effect of divalent metal ions on the activity of glycerophosphocholine cholinephosphodiesterse from ox brain was examined. Zn²⁺- and Co²⁺-glycerophosphocholine cholinephosphodiesterases were prepared from the exposure of apoenzyme to Zn²⁺ and Co²⁺, respectively, and the properties of two metallo-phosphodiesterases were compared to those of native phosphodiesterase. Although two metallo-enzymes were similar in expressing Km value, optimum pH or sensitivity to Cu²⁺, they differed in the susceptibility to the inhibition by thiocholine or tellurite; while Co²⁺-phosphodiesterase was more sensitive to tellurites, Zn²⁺-phosphodiesterase was more susceptible to inhibition by thiocholine. In addition, Zn²⁺-phosphodiesterase was more thermo-stable than Co²⁺ enzyme. Separately, when properties of native phosphodiesterase were compared to those of each metallo-phosphodiesterase, native phosphodiesterase was found to be quite similar to Zn²⁺-phosphodiesterase in many respects. Even in thermo-stability, native enzyme resembled Zn²⁺-phosphodiesterase rather than Co²⁺-enzyme. Consistent with this, the stability of native phosphodiesterase was maintained in the presence of Zn²⁺, but not Co²⁺. Mn²⁺ was also as effective as Zn²⁺ in the stabilization of the enzyme. Noteworthy, the native enzyme was found to be inhibited competitively by Cu²⁺ with a Ki value of 20 M, and its inhibitory action was antagonized effectively by Zn²⁺ or Co²⁺. Also, choline, another competitive inhibitor of the enzyme, appeared to antagonize the inhibitory action of Cu²⁺. Taken together, it is suggested that there may be multiple binding sites for divalent metal ions in the molecule of glycerophosphocholine cholinephosphodiesterase.     

Enzymatic Release of Zn2+-Glycerophosphocholine Cholinephosphodiesterase from Brain Membranes by Glycosylphosphatidylinositol-Specific Phospholipases and its Regulation

Enzymatic conversion of glycosylphosphatidylinositol (GPI)-linked Zn²⁺-glycerophosphocholine phosphodiesterase was investigated. The activity of glycosylphosphatidylinositol-specific phospholipase-D (GPI-PLD), based on the conversion of amphiphilic form of phosphodiesterase into hydrophilic form, showing an optimum pH of about pH 6.6, increased continuously until 60 min. The activity of membrane-bound GPI-PL, based on the formation of hydrophilic form of phosphodiesterase, exhibiting an optimum pH of 7.4, increased up to 30 min, and reached a plateau. Inhibition studies indicate that while GPI-PLD activity was generally sensitive to ionic bio-detergents, it was not inhibited by myristoyl glycerol, a neutal detergent. Meanwhile, the membrane-bound GPI-PL was not affected remarkably by these detergents except that myristoyl glycerol expressed a modest increase of activity of membrane bound GPI-PL. In addition, the membrane-bound GPI-PL appeared to be enhanced by by suramin or oleic acid, which strongly inhibited GPI-PLD. From this results, it is suggested that in brain there may be two phospholipases responsible for the conversion of membrane-bound GPI-anchors to hydrophilic forms, and that this conversion might be regulated by endogenous lipids.     

Release of GPI-Anchored Zn2+-Glycerophosphocholine Cholinephosphodiesterase as an Amphiphilic Form from Bovine Brain Membranes by Bee Venom Phospholipase A2

Enzymatic release of Zn²⁺-glycerophosphocholine (GPC)cholinephosphodiesterase, as an amphiphilic form, from bovine brain membranes was examined. Of various membrane hydrolases, bee PLA₂ was the most effective in the release of the GPC cholinephosphodiesterase (amphiphilic form, 63–70%) from membrane. Compared to pancreatic PLA₂, bee PLA₂ was more efficient in the release of GPC cholinephosphodiesterase. In pH-dependent release of GPl-anchored phosphodiesterase, there was a similar pH-release profile between PLA₂-mediated release and spontaneous one, implying the involvement of membrane disruption in the PLA₂ action. The PLA₂-mediated release showed a limited time-dependence (until 45 min) and a limited dose dependence (up to 3 units / ml), characteristic of a receptor-type binding. An ionic binding of PLA₂ to membrane may be alluded from the interfering effect of anionic phospholipids on the PLA₂ action. In support of an interaction between PLA₂ and membrane glycoproteins, the PLA₂ action was found to be blocked by lectins, wheat germ agglutinin or concanavalin A. In combination with detergent, the PLA₂-mediated release was found to be enhanced synergistically by saponin, a cholesterol-complexing agent. Meanwhile, an additive interaction between PLA₂ and lysolecithin suggests that PLA₂ action is independent of lysolecithin. It is suggested that the binding of PLA₂ to specific sites of membranes, probably rich in GPI-anchored glycoproteins, may be related to the facilitated release of GPI-anchored proteins as amphiphilic form.     

Involvement of both a Zn2+ site and an anionic binding site in the selective inhibition of a Zn2+-glycerophosphocholine cholinephosphodiesterase by thiols and tellurites

Inhibition of a Zn²⁺-glycerophosphocholine cholinephosphodiesterase by thiols or tellurites were examined mechanistically. Inactivation of the phosphodiesterase by thio-carboxylates, which was due to the removal of Zn²⁺ in the catalytic site, was enhanced by introduction of an amino group in the structure of thiols, suggesting the presence of an anionic site adjacent to a Zn²⁺ site. In support of the suggestion, it was found that thiols, associable with both a Zn²⁺ site and an anionic site, were more potent reversible inhibitors; dimethylaminoethanethiol (Ki, 17 M), diethylaminoethanethiol (Ki, 1.2 M) and thiocholine (Ki, 2.6 M). Meanwhile, the inhibition of the phosphodiesterase by tellurites is ascribed to the binding of tellurite anions to a Zn²⁺ site, based on the protective action of tellurite anions against the inactivation of the enzyme by EDTA. Moreover, the inhibition of the phosphodiesterase by tellurites was prevented by phosphate ions, which expressed the protective effect against EDTA inactivation. In further support, it was observed that gellurite and thiocholine appeared to interact with active site in an additive manner, in contrast to a synergistic action between tellurites and quaternary ammonium compounds such as acetylcholine or choline.     

Ascorbate-Induced Oxidative Inactivation of Zn2+-Glycerophosphocholine Cholinephosphodiesterase

Abstract: Zn²⁺-glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38°C. The inclusion of copper ions or Fe²⁺ accelerated the ascorbate-induced inactivation, with Cu²⁺ or Cu⁺ being much more effective than Fe²⁺, suggestive of ascorbate-mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H₂O₂ inactivated the enzyme in a concentration-dependent manner. Also, the enzyme was inactivated partially by a superoxide anion-generating system but not an HOCl generator. In support of involvement of H₂O₂ in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous ^•OH. Although p -nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu²⁺-induced inactivation of the phosphodiesterase was compared with the ascorbate-mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu²⁺ action. Here, it is proposed that Zn²⁺-glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.     

Brain myelin-bound Zn2+-glycerophosphocholine cholinephosphodiesterase is a glycosylphosphatidylinositol-anchored enzyme of two different molecular forms

A Zn²⁺-GPC cholinephosphodiesterase activity, which is present more predominently in myelin than in microsome or cytosol, has been examined using -nitrophenylphosphocholine as a substrate. In the solubilization of enzyme activity from myelin membranes, lysolecithin was found to be more effective than Triton X-100 or deoxycholate. Especially, the myelin-bound phosphodiesterase was suggested to be a glycosylphosphatidyl-inositol-anchored protein, based on solubilization by B. cereus phospholipase C and Triton X-114 phase separation. Interestingly, it was found that while phospholipase C-solubilized enzyme, a hydrophilic protein, was associable with Concanavalin A column, detergent-solubilized amphiphilic form of enzyme was not. Either detergent extract or cytosol was observed to contain both amphiphilic form and hydrophilic one. In CM-sephadex chromatography, the soluble hydrophilic phosphodiesterase was observed to be separatable into two forms of enzyme. In comparative studies, both forms of phosphodiesterase showed much similarity in substrate specificity, optimum pH, Km value and Zn²⁺ requirement, although they differed in charge property and molecular weight.     

Regulation and Prediction of Enzyme Activity in Reversed Micelles

The rate of cholesterol oxidation has been studied in cholesterol oxidase containing reversed micellar media consisting of the surfactant cetyltrimethylammonium bromide (CTAB), the surfactant octanol, a buffered aqueous solution, and a variety of organic solvents. By varying the composition of the medium systematically it could be deduced that the rate of cholesterol oxidation obeys the same rules as described earlier for the conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase in CTAB-hexanol-organic solvent reversed micelles (Hilhorst et al. 1984). The general applicability of these rules in optimizing biocatalysis in reversed micelles is discussed.     

Probing the Structural Basis of Zn2+ Regulation of the Epithelial Na+ Channel

Extracellular Zn²⁺ activates the epithelial Na⁺ channel (ENaC) by relieving Na⁺ self-inhibition. However, a biphasic Zn²⁺ dose response was observed, suggesting that Zn²⁺ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn²⁺, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn²⁺ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn²⁺ activating site, γH239A diminished Na⁺ self-inhibition and thus concealed the activating effects of Zn²⁺. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn²⁺ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp⁵¹⁶ was predicted to be in close proximity to γHis⁸⁸. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn²⁺. Our results suggest that external Zn²⁺ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His¹⁹³, His²⁰⁰, and His²⁰² and (ii) a low-affinity Zn²⁺ inhibitory site within the palm subdomain that includes His⁸⁸ and Asp⁵¹⁶.     

Molecular Dynamics Simulation of Substrate-Enzyme Interactions in the Active Site Channel of Superoxide Dismutase

Abstract

Molecular dynamics simulations of the diffusion of superoxide ion down the active site channel of the enzyme superoxide dismutase were performed with a parallelized version of GROMOS on the Intel iPSC/860. Our model consisted of a spherical assembly of 6968 atoms centered at a copper ion in the enzyme. Trajectory analysis revealed that the anion is directed toward the copper ion through the cooperative motions of several active site residues. Other mechanistic and structural motifs recurring through five full trajectories are examined. In addition to these qualitative results, an upper bound has been calculated for the rate constant for displacement by substrate of the water molecule that is coordinated to the copper. This required an analysis of the dynamics of crossing a free energy barrier that has been characterized in previous work. Strong frictional effects due to Coulombic interactions lead to a rather small rate constant; the transmission coefficient is less than 0.01. The mechanism of the enzyme therefore may involve diffusion of substrate up to the bound water followed by electron transfer mediated by this water, rather than displacement of the water by substrate with subsequent electron transfer.     

The Low Affinity Dopamine Binding Site on Tyrosine Hydroxylase: The Role of the N-Terminus and In Situ Regulation of Enzyme Activity

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.     

Activation of sphingomyelinase from Bacillus cereus by Zn2+ hitherto accepted as a strong inhibitor

Sphingomyelinase (SMase) from Bacillus cereus has been known to be activated by Mg2+, Mn2+, and Co2+, but strongly inhibited by Zn2+. In the present study, we investigated the effects of several kinds of metal ions on the catalytic activity of B. cereus SMase, and found that the activity was inhibited by Zn2+ at its higher concentrations or at higher pH values, but unexpectedly activated at lower Zn2+ concentrations or at lower pH values. This result indicates that SMase possesses at least two different binding sites for Zn2+ and that the Zn2+ binding to the high-affinity site can activate the enzyme, whereas the Zn2+ binding to the low-affinity site can inactivate it. We also found that the binding of substrate to the enzyme was independent of the Zn2+ binding to the high-affinity site, but was competitively inhibited by the Zn2+ binding to the low-affinity site. The binding affinity of the metal ions to the site for activating the enzyme was determined to be in the rank-order of Mg2+ = Co2+ < Mn2+ < Zn2+. It was also demonstrated that these four metal ions competed with each other for the same binding site on the enzyme molecule.     

Aspergillomarasmine A inhibits metallo-β-lactamases by selectively sequestering Zn2+

Class B metallo-β-lactamases (MBLs) are Zn²⁺-dependent enzymes that catalyze the hydrolysis of β-lactam antibiotics to confer resistance in bacteria. Several problematic groups of MBLs belong to subclass B1, including the binuclear New Delhi MBL (NDM), Verona integrin-encoded MBL, and imipenemase-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarasmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. The precise mechanism of action of AMA is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn²⁺ cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn²⁺ scavenger that indirectly inactivates NDM-1 by encouraging the dissociation of a metal cofactor. To further investigate the mechanism in living bacteria, we used an active site probe and showed that AMA causes the loss of a Zn²⁺ ion from a low-affinity binding site of NDM-1. Zn²⁺-depleted NDM-1 is rapidly degraded, contributing to the efficacy of AMA as a β-lactam potentiator. However, MBLs with higher metal affinity and stability such as NDM-6 and imipenemase-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn²⁺ chelators and highlight that leveraging Zn²⁺ availability can influence the survival of MBL-producing bacteria when they are exposed to β-lactam antibiotics.     

邻苯二甲醛修饰法探测中华猕猴桃蛋白酶在胍溶液中活性部位的构象变化

Palczewsski等［1］以邻苯二甲醛修饰醛缩酶活性部位的氨基和流基以形成一异蚓噪环,利用该基团的荧光特性来探测醛缩酶的活性部位构象,Weq［2］,Le［3］并成功地运用这一方法研究肌酸激酶和酵母乙醇脱氢酶的活性部位构象变化．中华猕猴桃蛋白酶的唯一游离流基（CyS-25）是催化功能团【'」,而且氨基也是活性部位的必需基因【到,符合邻苯二甲醛的反应性,所以我们借鉴Pal＿ski等的方法【1］将这一荧光基因引人中华猕猴桃蛋白酶,用以探测该酶在抓溶液中活性部位的构象变化,并与相应的活力变化以及酶的内源荧光及CD谱变化作比较．1材…     

Active Site Studies on a Narrow-Specificity Thyroliberin-Hydrolysing Pyroglutamate Aminopeptidase Purified from Synaptosomal Membrane of Guinea-Pig Brain

The effect of protein-modifying reagents on the activity of a purified preparation of a thyroliberin-hydrolysing pyroglutamate aminopeptidase, solubilised from synaptosomal membranes of guinea-pig brain by treatment with papain, was investigated. The results indicated that tyrosine, histidine, arginine, and possibly lysine residues were necessary for expression of catalytic activity and that these tyrosine, histidine, and arginine residues were probably located at the active site of the enzyme. Cysteine, serine, glutamate, and aspartate residues were not involved in the expression of catalytic activity.     

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn²⁺ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn²⁺ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp⁵⁹⁹ and His⁴⁵¹, with alanine was sufficient to block Zn²⁺ transport. These findings indicate, for the first time, that Zn²⁺ transport mediated by a mammalian ZnT is catalyzed by H⁺/Zn²⁺ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.     

成熟香蕉果实活性氧与乙烯形成酶活性的关系

香蕉果实成熟过程,随着活性氧产生速率从低水平→迅速跃升→高峰→下降的变化,其乙烯形成酶活性及乙烯产生也经历了基本同步的过程,显示了三者之间具有某种内在联系。外源超氧阴离子自由基（O2）能使乙烯形成酶（EFE）活性及乙烯产生出现跃升和高峰的时间明显提前;超氧歧化酶（SOD）则使EFE活性及乙烯产率明显下降。进一步说明了在香蕉果实成熟过程中,O2可能是引起EFE活性及乙烯产生迅速上升的原因之一,而过氧化氢（H2O2）则被证明与EFE活性及乙烯产生没有直接的关系。     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.     

Competition of Mn2+ and Zn2+ with59Fe2+ and59Fe3+ for the plasma membrane receptors from lactating mouse mammary gland

Nonlabeled MnCl₂ and ZnSO₄ compete with⁵⁹Fe²⁺-ascorbate and⁵⁹Fe ₂ ³⁺ O₃ for transport binding sites situated on the plasma membranes of lactating mouse mammary gland cells. The binding was found to be a process reaching saturation. The heterologous competition used here ruled out the participation of transferrin and to propose that Fe, Mn, and Zn are transported from blood to milk by a mechanism involving one receptor during lactation. Further experiments are necessary to establish the details of the transport mechanism.     

Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery.     

Regulation of protein kinase C activity by cooperative interaction of Zn2+ and Ca2+

cis-Fatty acids such as oleic acid or linoleic acid have been previously shown to induce full activation of protein kinase C in the absence of Ca2+ and phospholipids (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193; Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429). In this study, we have investigated the effects of various metal ions on protein kinase C activity without the interference of Ca2+ since cis-fatty acid requires no Ca2+ for protein kinase C activation. Here we report a specific interaction of Zn2+ with protein kinase C in either a positive or negative cooperative fashion in concert with Ca2+. At low concentrations (approximately 5 microM) of Ca2+, Zn2+ enhances protein kinase C activity induced by both oleic acid and phosphatidylserine/diolein. In contrast, Zn2+ inhibits the activity at higher concentrations (over 50 microM) of Ca2+. In the absence of Ca2+, Zn2+ shows no effect on protein kinase C activity. Our results suggest that Zn2+ does not recognize or interact with protein kinase C in the absence of Ca2+, that protein kinase C possesses high and low affinity Ca2+-binding sites, and that at least one Zn2+-binding site exists which is distinct from Ca2+-binding sites.