Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule.     

Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.     

Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Spectroscopic studies on the metal-ion-binding sites of Co2(+)-substituted D-xylose isomerase from Streptomyces rubiginosus

The coordination sphere of the two metal-binding sites/subunit of the homotetrameric D-xylose isomerase from Streptomyces rubiginosus has been probed by the investigation of the Co2(+)-substituted enzyme using electronic absorption, CD and magnetic circular dichroic spectroscopies in the visible region. The spectrum of the high-affinity site (B site) has an absorption coefficient, epsilon 545, of 18 M-1 cm-1, indicating a distorted octahedral complex geometry. The spectrum of the low-affinity site (A site) shows two absorption maxima at 505 nm and 586 nm with epsilon values of 170 M-1 cm-1 and 240 M-1 cm-1, respectively, which indicates a distorted tetrahedral or pentacoordinated complex structure as also observed for the enzyme from Streptomyces violaceoruber [Callens et al. (1988) Biochem. J. 250, 285-290] having the same feature but lower epsilon values. The first 4 mol Co2+ added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co2+/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd2+ or Pb2+/mol Co4-loaded derivative displaces the Co2+ from the B site to form the Pb4/Co4 derivative containing Co2+ in the A site, reducing activity fourfold while the Pb4/Pb4 species is completely inactive. In contrast, Eu3+ displaces Co2+ preferentially from the A site. Thus, the high- and low-affinity sites may be different for different cations. After addition of the substrates D-xylose, D-glucose and D-fructose and the inhibitor xylitol the intense Co2+ A-site spectrum of both the active Co4/Co4 derivative and the less active Pb4/PCo4 derivative decreases, indicating that these compounds are bound to the A site, changing the distorted tetrahedral or pentacoordinated symmetry there to a distorted octahedral complex geometry.     

Metal ion binding to alpha-lactalbumin species

A strong cation (calcium) binding site has been demonstrated to exist in several alpha-lactalbumin species; bovine, goat, human, and guinea pig. A metal ion induced conformational change occurs, resulting in a unique (10-14-nm) blue shift and relative quenching of Trp fluorescence for all species. Calcium ion binding to the alpha-lactalbumins yielded dissociation constants (Kdiss consistently in the 10(-10)--10(-12) M range, while Mn(II) binding was in the 20-30 microM range. Independent determinations of these cation binding equilibria were made by ESR measurements of free unliganded Mn(II) in titrations with the bovine species. One strong site (Kdiss = 30.5 microM) was found, which correlated directly with the fluorescence-associated cation binding, plus three weaker sites (Kdiss = 1.1, 5.0, and 5.0 mM, respectively). Several lanthanides as well as Mg(II) were found to displace Mn(II) from the strong site on bovine alpha-lactalbumin (as monitored by ESR) and to cause the identical fluorescence changes as found for Ca(II) and Mn(II) above. The importance of measuring these equilibria by both fluorescence and ESR was borne out by demonstrating the potential errors in estimating dissociation equilibria by the fluorescence method alone. Also, the errors in estimating Kdiss for samples containing partially metal bound apo-alpha-lactalbumin are described as well as rapid, sensitive methods for estimating the extent of metal-free protein and correctly accounting for residual bound metal in equilibrium calculations.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献   

Metal ion binding to concanavalin A

Metal ion activation of saccharide binding has been studied for concana-valin A near pH 7.0. Although two metal ions, a transition metal ion and a Ca²⁺ ion, can bind, both are not required. Ca²⁺ alone, Mn²⁺ alone, or Ca²⁺ with other transition metal ions can activate this lectin. Only one Ca²⁺ ion per subunit or only one Mn²⁺ per subunit is sufficient. Metal ion binding was studied by magnetic resonance techniques and direct binding assays. Saccharide binding activity was monitored by following the fluorescence of 4-methylumbelliferyl a-D-mannopyranoside. When Ca²⁺ binds to demetalized concanavalin A, the transition metal ion site is hindered. When Mn²⁺ alone binds to demetalized concanavalin A, saccharide binding activity is induced. A subsequent conformational change, not necessary for carbohydrate binding activity, covers the Mn²⁺.     

Extraction of glucose isomerase from Streptomyces flavogriseus.

Cationic detergent (cetyltrimethylammonium bromide or cetylpyridinium chloride) treatment extracted almost the same amount of glucose isomerase from cells of Streptomyces flavogriseus as mechanical disruption (sonic oscillation or abrasive grinding). The specific activity of the enzyme extracted with cationic detergents was approximately 20% higher than that liberated by mechanical disruption.     

Metal ion binding in triclinic lysozyme.

The binding sites of Mn2+, Co2+, and Gd3+ have been determined in triclinic lysozyme at pH 4.5 to 4.6. Mn2+ and Co2+ bind a site approximately 2.5 A from 1 of the oxygen atoms of the Glu-35 chain. The occupancy of the Mn2+ site is 0.22, corresponding to 1 bound ion for each 4.6 protein molecules. The occupancy of the Co2+ site is much lower, about 0.048. Gd3+ appears to be bound at two sites, the main one 2.5 A from an oxygen atom of the Glu-35 side chain, the other 3.1 A from an oxygen atom of the Asp-52 chain. The occupancy of both Gd3+ sites is low, 0.036 and 0.016, the latter being so low that the presence of the ion at this site is in doubt. The binding site of Mn2+ in the di(N-acetylglucosamine)-lysozyme complex has also been determined. It does not differ significantly from the Mn2+ binding site in the native protein, but the occupancy is lower, 0.16.     

Studies on glucose isomerase from a Streptomyces species.

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.     

Fermentation of D-xylose by yeasts using glucose isomerase in the medium to convert D-xylose to D-xylulose

Summary A method to obtain the fermentative conversion by yeasts of D-xylose to ethanol is described. The method depends on a combination of two factors; (1) the ability of glucose isomerase to isomerise D-xylose to D-xylulose and (2) the ability of a number of yeasts to ferment D-xylulose.     

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.     

Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.     

Metal ion and substrate binding to bovine galactosyltransferase

Bovine milk galactosyltransferase was examined by ESR and NMR proton relaxation measurements to determine the stoichiometry and nature of manganese and UDP-Gal substrate binding. The ESR and NMR data clearly showed the binding of two (Mn(II) per mol of enzyme in the ternary complex (enzyme-manganese-UDP-Gal). The affinity of the enzyme for manganese is much higher in the presence of UDP-Gal than in its absence. A deenhancement was observed in both water and UDP-Gal proton relaxation rates upon ternary complex formation [enzyme-Mn(II)-UDP-Gal] relative to the metal-substrate [Mn(II)-UDP-Gal] binary complex, yet the temperature dependence of the water proton relaxation rate was consistent with fast exchange. A simple model was proposed which accounted for the pronounced deenhancement, involving a slow conformational interconversion of an initially formed, rapidly exchanging conformer of the enzyme-Mn(II)-UDP-Gal complex to a second form which contributes negligibly to the relaxation.     

Kinetic properties and related changes of molecular weight in a fructokinase from Streptomyces violaceoruber.

1. A study of the initial reaction rates at variable substrate concentrations and of the molecular weight of the enzyme in the presence of different effectors, has been carried out using fructokinase (ATP: fructose 6-phosphotransferase, EC 2.7.1.4) from Streptomyces violaceoruber. 2. Saturation curves for MgATP or CoATP are sigmoidal and they change to hyperbolic in the presence of 10 mM Mg2+ or Co2+ in excess over the nucleoside triphosphate. 3. Saturation cuvves for fructose show intermediary plateaux at high (but not at low) concentrations of ATP or Mg2+. 4. The molecular weight of the enzyme in the presence of high concentrations of MgATP is 80 000. In the presence of fructose, and/or Mg2+, the molecular weight is 20 000. 5. The effects of MgADP, uncomplexed ADP or ATP, and low concentrations of detergent on the kinetics have been studied. The results are interpreted as showing the existence of cooperative effects.     

Purification of glucose isomerase from Streptomyces nigrificans

Summary A relatively simple method for obtaining an electrophoretically homogeneous preparation of glucose isomerase from Streptomyces nigrificans is described. Extract of disintegrated microbial cells was first heated at 60°C in the presence of Co and Mg ions. Centrifugation and ultrafiltration were followed by ion exchange chromatography on DEAE-cellulose. The fraction with glucose isomerase activity proved to contain no proteins other than the isolated enzyme.