一些理化因子对α淀粉酶构象与活力的影响

用荧光光谱,紫外差示光谱和ＣＤ谱研究了一些理化因子对枯草芽孢杆菌８６３１５α淀粉酶的构象与活力的影响,实验表明,酸变性和碱变性所引起的酶构象变化是不同的;乙醇不降低α淀粉酶活力,但使其构象发生较大变化,α螺旋度从天然酶的２６,１％降到２１．８％,其构象变化不引起活性中心的改变;酶在７０℃处理１０ｍｉｎ后,由原来紧密构象变为松散构象,α螺旋度从２６．１％降到９．０％,酶活性完全丧失;而在０．０２ｍｏｌ／ＬＣａＣｌ_２和０．０２ｍｏｌ／ＬＮａＣｌ的共同存在下,７０℃处理１０ｍｉｎ,酶活性不变,其荧光光谱和ＣＤ谱接近于天然酶,所以,ＣａＣ_ｌ２和ＮａＣｌ能保护α淀粉酶的构象,使之不受热变性。     

Reversible inactivation of cytochrome P450 by alkaline earth metal ions: auxiliary metal ion induced conformation change and formation of inactive P420 species in CYP101

The effects of the divalent alkaline-earth metal ions (Ca²⁺ and Mg²⁺) on the substrate binding affinity, spin-state transition at the heme active site, conformational properties as well as the stability of the active form of cytochrome P450cam (CYP 101) have been investigated using various spectroscopic and kinetic methods. The divalent cations were found to have two types of effects on the enzyme. At the initial stage the alkaline-earth metal ion facilitated enhanced binding of the substrate and formation of the high-spin form of the heme active center of the enzyme compared to that in absence of any metal ion. However, analogous to many other mono-valent metal ions, the alkaline-earth metal ions were also less efficient than K⁺ in promoting the substrate binding and spin-transition properties of the enzyme. The auxiliary metal ions were shown to cause small but distinct change in the circular dichroism spectra of the substrate-free enzyme in the visible region, indicating that the tertiary structure around the heme was perturbed on binding of the auxiliary metal ion to the enzyme. The effect of the auxiliary metal ion was found to be more prominent in the WT enzyme compared to the Y96F mutant of P450cam suggesting that the Tyr 96 residue plays an important role in mediating the effects of the auxiliary metal ions to the active site of the enzyme. At the second stage of interaction, the alkaline-earth metal ions were found to slowly convert the enzyme into an inactive P420 form, which could be reversibly re-activated by addition of KCl. The results have been discussed in the light of understanding the mechanism of inactivation of certain mammalian P450 enzymes by these alkaline-earth metal ions.     

Circular dichroism (CD) studies on yeast enolase: activation by divalent cations

The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes. This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure. There was no effect of magnesium ion on the far ultraviolet spectrum. Evidently magnesium ion has no effect on the secondary structure. Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity. The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions. Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes. The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups. Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme. The "catalytic" and "inhibitory" sites did not appear to be very CD active.     

Muscle contraction: viscous-like frictional forces and the impulsive model

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.     

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.     

Calcium-ion-induced stabilization of the protease from Bacillus cereus WQ9-2 in aqueous hydrophilic solvents: effect of calcium ion binding on the hydration shell and intramolecular interactions

The neutral protease WQ from Bacillus cereus is stable in various aqueous organic mixtures, with the exception of those containing acetonitrile (ACN) and dimethylformamide (DMF). The stability of the enzyme in aqueous hydrophilic solvents was dramatically enhanced with the addition of calcium ions, with the degree of improvement in the half-life relative to different solutions ranging from fourfold to more than 70-fold. Studies of the kinetic constants showed that calcium ions induced slight conformational changes in the active site of the enzyme in aqueous ACN. We investigated the molecular mechanisms underlying this stabilizing effect by employing a combination of biophysical techniques and molecular dynamics simulation. In aqueous ACN, the intrinsic fluorescence and circular dichroism analysis demonstrated that the addition of calcium ions induced a relatively compact conformation and maintained both the native-like microenvironment near the tryptophan residues and the secondary structure. Alternatively, homology modeling confirmed the location of four calcium-ion-binding sites in the enzyme, and molecular dynamics simulation revealed that three other calcium ions were bound to the surface of the enzyme. Calcium ions, known as a type of kosmotrope, can strongly bond with water molecules, thus aiding in the formation of the regional hydration shell required for the maintenance of enzyme activity. In addition, the introduction of calcium ions resulted in the formation of additional ionic interactions, providing propitious means for protein stabilization. Thus, the stronger intramolecular interactions were also expected to contribute partially to the enhanced stability of the enzyme in an aqueous organic solvent.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Enhancing effect of calcium and vanadium ions on thermal stability of bromoperoxidase from Corallina pilulifera

Bromoperoxidase from the macro-alga Corallina pilulifera is an enzyme that possesses vanadate in the catalytic center, and shows a significant thermostability and stability toward organic solvents. The structural analysis of the recombinant enzyme overexpressed in yeast revealed that it contains one calcium atom per subunit. This has been confirmed by inductively coupled plasma emission spectrometry experiments. The study of the effect of metal ions on the apo-enzyme stability has shown that the calcium ion significantly increased the enzyme stability. In addition, vanadate also increased the thermostability and strontium and magnesium ions had similar effects as calcium. The holo-enzyme shows high stability in a range of organic solvents. The effect of the different ions and solvents on the structure of the enzyme has been studied by circular dichroism experiments. The high stability of the enzyme in the presence of organic solvents is useful for its application as a biocatalyst.     

Slow binding of metal ions to pigeon liver malic enzyme: a general case

Pigeon liver malic enzyme was inhibited by lutetium ion through a slow-binding process, which resulted in a concave down tracing of the enzyme activity assay. The fast initial rates were independent of lutetium ion concentration, while the slow steady-state rates decreased with increasing Lu(3+) concentration. The observed rate constant for the transition from initial rate to steady-state rate, k(obs), exhibited saturation kinetics as a function of Lu(3+) concentration, suggesting the involvement of an isomerization process between two enzyme forms (R-form and T-form). The binding affinity of Lu(3+) to the R-form is weaker (K(d,Lu) = 14 microM) than that of Mn(2+) (K(m,Mn) = 1.89 microM); however, Lu(3+) has much tighter binding affinity with the T-form ( = 0.83 microM). Lu(3+) was shown to be a competitive inhibitor with respect to Mn(2+), which suggests that Lu(3+) and Mn(2+) are competing for the same metal binding site of the enzyme. These observations are in accordance with the available crystal structure information, which shows a distorted active site region of the Lu(3+)-containing enzyme. Other divalent cations, i.e., Fe(2+), Cu(2+), or Zn(2+), also act as time-dependent slow inhibitors for malic enzyme. The dynamic quenching constants of the intrinsic fluorescence for the metal-free and Lu(3+)-containing enzymes are quite different, indicating the conformational differences between the two enzyme forms. The secondary structure of these two enzyme forms, on the other hand, was not changed. The above results indicated that replacement of the catalytically essential Mn(2+) by other metal ions leads to a slow conformational change of the enzyme and consequently alters the geometry of the active site. The transformed enzyme conformation, however, is unfavorable for catalysis. Both the chemical nature of the metal ion and its correct coordination in the active site are essential for catalysis.     

Metal-induced structural organization and stabilization of the metalloregulatory protein MntR

MntR is a metalloregulatory protein that helps to modulate the level of manganese in Bacillus subtilis. MntR shows a metal-response profile distinct from other members of the DtxR family of metalloregulatory proteins, which are generally considered to be iron(II)-activated. As part of an ongoing effort to elucidate the mechanism and metal-selectivity of MntR, several biophysical studies on wild-type MntR and two active site mutants, MntR E99C and MntR D8M, have been performed. Using circular dichroism (CD) spectroscopy, the thermal stability of these proteins has been examined in the presence of various divalent metal ions. Fluorescence intensity measurements of 8-anilino-1-naphthalenesulfonic acid (ANS) were monitored to examine the folding of these proteins in the presence of different metal ions. These experiments indicate that MntR undergoes a significant conformational change upon metal binding that results in stabilization of the protein structure. These studies also show that the MntR D8M active site mutation causes a detrimental effect on the metal-responsiveness of this protein. Fluorescence anisotropy experiments have been performed to quantify the extent of metal-activated DNA binding by these proteins to two different cognate recognition sequences. Binding of MntR and MntR E99C to the mntA cognate sequence closely parallels that of the mntH operator, confirming that the proteins bind both sequences with comparable affinity depending on the activating metal ion. Fluorescence anisotropy experiments on MntR D8M indicate significantly impaired DNA binding, providing additional evidence that MntR D8M is a dysfunctional regulator.     

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.     

Effect of ionic strength and oxidation on the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr

The protein tyrosine phosphatase (PTP)-like phytase, PhyAsr, from Selenomonas ruminantium is a novel member of the PTP superfamily, and the only described member that hydrolyzes myo-inositol-1,2,3,4,5,6-hexakisphosphate. In addition to the unique substrate specificity of PhyAsr, the phosphate-binding loop (P-loop) has been reported to undergo a conformational change from an open (inactive) to a closed (active) conformation upon ligand binding at low ionic strength. At high ionic strengths, the P-loop was observed in the closed, active conformation in both the presence and absence of ligand. To test whether the P-loop movement can be induced by changes in ionic strength, we examined the effect that ionic strength has on the catalytic efficiency of PhyAsr, and determined the structure of the enzyme at several ionic strengths. The catalytic efficiency of PhyAsr is highly sensitive to ionic strength, with a seven-fold increase in k(cat)/K(m) and a ninefold decrease in K(m) when the ionic strength is increased from 100 to 500 mm. Surprisingly, the P-loop is observed in the catalytically competent conformation at all ionic strengths, despite the absence of a ligand. Here we provide structural evidence that the ionic strength dependence of PhyAsr and the conformational change in the P-loop are not linked. Furthermore, we demonstrate that the previously reported P-loop conformational change is a result of irreversible oxidation of the active site thiolate. Finally, we rationalize the observed P-loop conformational changes observed in all oxidized PTP structures.     

Characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase

The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg(2+) or Mn(2+) ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K(d) values for the binding of Mg(2+) and Mn(2+) ions to the free enzyme were 3.1 and 0.3 mm, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ion-induced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state.     

The effects of metal ions on the structure and stability of the DNA gyrase B protein

The effects of mono- and divalent metal ions on the DNA gyrase B subunit, on its 43 kDa and 47 kDa domains, and on two mutants in the Toprim domain (D498A and D500C) were investigated by means of circular dichroism and protein melting experiments. Both types of metal ion, with the notable exception of Mn2+, did not affect the conformational properties of the enzyme subunit at room temperature, but were able to produce selective and differential effects on protein stability. In particular, monovalent (K+) ions increased the stability of the gyrase B structure, whereas destabilising effects were most prominent using Mn2+ as the metal ion. Ca2+ and Mg2+ produced comparable changes in the gyrase B melting profile. Additionally, we found that monovalent (K+) ions were more effective in the 43 kDa N-terminal domain where ATP binding occurs, whereas divalent ions caused large modifications in the conformational stability of the 47 kDa C-terminal domain. Our results on gyrase B mutants indicate that D498 interacts with Mn2+, whereas it has little effect on the binding of the other ions tested. A D500C mutation, in contrast, effectively impairs Mg2+ affinity, suggesting effective contacts between this ion and D500 in the wild-type enzyme. Hence, the sites of metal ion complexation within the Toprim domain are modulated by the nature of the ion species. These results suggest a double role played by metal ions in the catalytic steps involving DNA gyrase B. One has to do with direct involvement of cations complexed to the Toprim domain in the DNA cutting-rejoining process, the other, until now overlooked, is connected to the dramatic changes in protein flexibility produced by ion binding, which reduces the energy required for the huge conformational changes essential for the catalytic cycle to occur.     

Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states

Phytases hydrolyze phytic acid to less phosphorylated myo-inositol derivatives and inorganic phosphate. A thermostable phytase is of great value in applications for improving phosphate and metal ion availability in animal feed, and thereby reducing phosphate pollution to the environment. Here, we report a new folding architecture of a six-bladed propeller for phosphatase activity revealed by the 2.1 A crystal structures of a novel, thermostable phytase determined in both the partially and fully Ca2+-loaded states. Binding of two calcium ions to high-affinity calcium binding sites results in a dramatic increase in thermostability (by as much as approximately 30 degrees C in melting temperature) by joining loop segments remote in the amino acid sequence. Binding of three additional calcium ions to low-affinity calcium binding sites at the top of the molecule turns on the catalytic activity of the enzyme by converting the highly negatively charged cleft into a favorable environment for the binding of phytate.     

变性过程中铜锌超氧化物歧化酶的活性通道

在酶的盐酸胍变性和热变性过程中,尝试采用电荷传递反应分析方法和电子自旋共振方法考察了酶活性部位的构象变化。酶活力与构象的变化行为表明,酶的活性部位通道先于酶分子的整体构象而发生变化,它是与酶的失活同时发生的。尽管酶活性部位中的金属离子保证了酶较高的稳定性,但酶的活性部位,特别是活性通道仍然是相对脆弱的。     

Structure of the complex of active site metal-depleted horse liver alcohol dehydrogenase and NADH.

The complex between active site-specific metal-depleted horse liver alcohol dehydrogenase and NADH has been studied with X-ray crystallographic methods to 2.9 A resolution. The electron density maps revealed that only the catalytic zinc ions are removed, whereas the non-catalytic zinc sites ae fully occupied. A gross conformational change in the protein induced by co-enzyme binding takes place in this enzyme species despite the absence of the metal ion in the catalytic center. This circumstance is of great importance in the understanding and further analysis of the trigger mechanisms operating during the conformation transition in alcohol dehydrogenase, since the catalytic center is located at the hinge region for a domain rotation in the subunit, and the metal atom is essential for catalysis. The overall protein structure is the same as that of an NADH complex of the native zinc enzyme and the co-enzyme is bound in a similar manner. The local structural changes observed are restricted to the empty metal binding site.     

Investigating the role of metal ions in the catalytic mechanism of the yeast RNA triphosphatase

The Saccharomyces cerevisiae RNA triphosphatase (Cet1) requires the presence of metal ion cofactors to catalyze its phosphohydrolase activity, the first step in the formation of the 5'-terminal cap structure of mRNAs. We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction. The association of Mg2+, Mn2+, and Co2+ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was then used to determine the apparent dissociation constants for these ions. Subsequent dual ligand titration experiments demonstrated that the metal ions bind to a common site, for which they compete. The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated. To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicate that binding of RNA, nucleotides, and metal ion cofactors does not lead to significant structural modifications of the Cet1 architecture. This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site. Finally, denaturation studies demonstrate that the metal ion cofactors can act by stabilizing the ground state binding of the phosphohydrolase substrate.     

pH-induced conformational transition in the soluble CuA domain of Paracoccus denitrificans cytochrome oxidase

The pH-induced conformational transition in the CuA domain of subunit II of cytochrome oxidase of Paracoccus denitrificans (PdII) has been investigated using various spectroscopic and stopped-flow kinetic methods. UV-visible absorption and circular dichroism studies showed that an increase in pH from 6 to 10 leads to a conformation change with pK(a) = 8.2 associated with the CuA site of the protein. The secondary structure of the protein was, however, shown to remain unchanged in these two conformational states. Thermal and urea-induced unfolding studies showed that the "low-pH" conformation is more stable compared to the "high-pH" conformation of the protein. Moreover, the overall stability of the protein was found to decrease on reduction of the metal centers in the low-pH form, while the oxidation state of the metal centers did not have any significant effect on the overall stability of the protein in the high-pH form. Stopped-flow pH-jump kinetic studies suggested that the conformational transition is associated with a slow deprotonation step followed by fast conformational equilibrium. The results are discussed in the light of understanding the pH-induced conformational change in the beta-barrel structure of the protein and its effect on the coordination geometry of the metal site.     

酵母乙醇脱氢酶胍变性时的失活与去折叠的比较研究

应用荧光发射光谱,圆二色光谱,二阶导数光谱和紫外差吸收光谱等监测手段,研究了酵母乙醇脱氢酶在胍溶液中的去折叠。比较不同盐酸胍浓度下酵母乙醇脱氢酶的失活与构象变化,实验表明酶的失活先于构象变化：在低浓度胍溶液中,构象尚未发生明显变化时,酶活几乎已经完全丧失。由上述结果可见,含有辅基金属离子Ｚｎ~（２＋）酶的活性部位较酶分子的整体结构也具有柔性。