Isotopic Exchange plus Substrate and Inhibition Kinetics of D-Xylose Isomerase Do Not Support a Proton-Transfer Mechanism (original) (raw)
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Biotechnology and Bioengineering, 2002
The conversion of glucose to fructose at elevated temperatures, as catalyzed by soluble and immobilized xylose (glucose) isomerases from the hyperthermophiles Thermotoga maritima (TMGI) and Thermotoga neapolitana 5068 (TNGI) and from the mesophile Streptomyces murinus (SMGI), was examined. At pH 7.0 in the presence of Mg2+, the temperature optima for the three soluble enzymes were 85°C (SMGI), 95° to 100°C (TNGI), and >100°C (TMGI). Under certain conditions, soluble forms of the three enzymes exhibited an unusual, multiphasic inactivation behavior in which the decay rate slowed considerably after an initial rapid decline. However, the inactivation of the enzymes covalently immobilized to glass beads, monophasic in most cases, was characterized by a first-order decay rate intermediate between those of the initial rapid and slower phases for the soluble enzymes. Enzyme productivities for the three immobilized GIs were determined experimentally in the presence of Mg2+. The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60°C and 70°C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg2+ occurred at 70°C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80°C and in the presence of Mg2+, productivities for the three enzymes ranged from 31 to 273. A simple mathematical model, which accounted for thermal effects on kinetics, glucose–fructose equilibrium, and enzyme inactivation, was used to examine the potential for high-fructose corn syrup (HFCS) production at 80°C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co2+ and Mg2+. In the presence of both cations, these enzymes showed the potential to catalyze glucose-to-fructose conversion at 80°C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55° to 65°C, but with the additional advantage of higher fructose concentrations. At 90°C, the estimated productivity for SMGI dropped to 200, whereas, for TNGI, lifetime productivities on the order of 1000 were estimated. Assuming that the most favorable biocatalytic and thermostability features of these enzymes can be captured in immobilized form and the chemical lability of substrates and products can be minimized, HFCS production at high temperatures could be used to achieve higher fructose concentrations as well as create alternative processing strategies. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 185–194, 2002.
Glucose Isomerase and Its Behaviour under Hydrogenation Conditions
Starch-starke, 1985
A survey is given of glucose isomerase, its sources, its mechanism of isomerization and its data and properties in three different immobilized forms. In addition, the effect of a number of parameters on the activity of immobilized glucose isomerase has been investigated, e. g. hydrogen pressure, Mg(I1) and Ca(II), transition metal ions, borate and sugar alcohols. Immobilized glucose isomerase remains sufficiently active under hydrogenation conditions to maintain D-glucose and D-fructose in equilibrium. D-Glucitol, in contrast to D-mannitol, has some inhibiting effect on the enzyme action. The D-glUCOSe/Dfructose equilibrium constant is independent of the total sugar concentration (between 0.2-2.2-M). Glucoseisornerase und ihr Verhalten unter Hydrierungsbedingungen. Es wird eine Ubersicht iiber Glucoseisomerase, ihre Herkunfte, ihren Isomerisierungsmechanismus sowie uber ihre Daten und Eigenschaften in drei verschiedenen immobilisierten Formen gegeben. Daruber hinaus wurde die Wirkung einer Anzahl yon Parametern auf die Aktivitat von immobilisierter Glucoseisomerase, z. B. Wasserstoffdruck, Mg(I1) und Ca(II), Ubergangsmetall-Ionen sowie Borat und Zuckeralkohole, untersucht. Immobilisierte Glucoseisomerase behalt unter Hydrierungsbedingungen genugend Aktivitat, um D-Glucose und D-Fructose im Gleichgewicht zu halten. D-Glucitol (D-Sorbitol) hat im Gegensatz zu D-Mannitol einen gewissen inhibierenden EinfluD auf die Enzymwirkung. Die D-Glucose/D-Fructose-Gleichgewichtskonstante ist unabhangig von der Gesamtzuckerkonzentration (0,2-2,2-M).
Biochemistry and Cell Biology, 2001
A thermostable D-xylose-glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by gamma-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE-Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 73-92% homology with xylose-glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60-85 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.
Galactose Mutarotase: pH Dependence of Enzymatic Mutarotation †
Biochemistry, 2003
Here we report pH dependence of kinetic parameters for the mutarotation of R-D-glucose catalyzed by galactose mutarotase (GalM) from Escherichia coli. The values of k cat and k cat /K m for the mutarotation of R-D-galactose were found to be 1.84 × 10 4 s-1 and 4.6 × 10 6 M-1 s-1 , respectively, at pH 7.0 and 27°C. The corresponding values for R-D-glucose were 1.9 × 10 4 s-1 and 5.0 × 10 5 M-1 s-1. Inasmuch as the value of k cat /K m for the reaction of R-D-galactose is 10 times that for R-D-glucose, and the diffusional rate constants should be essentially the same for the two sugars, the mutarotation of R-Dglucose should not be diffusion controlled. Therefore, pH-rate profiles should not be distorted by diffusion. The k cat for the mutarotation of R-D-glucose is independent of pH. Therefore, either the enzyme-substrate complexes do not undergo ionization of catalytic groups, or the rate-limiting step is neither mutarotation nor diffusion. The profile of log k cat /K m versus pH is a distorted bell-shaped curve, with slopes of +1 on the acid side and-2 on the alkaline side. The values of pK a are 6.0 and 7.5, and mutarotation depends on the ionization states of three functional groups in the free enzyme, one unprotonated and two protonated. On the acid side, ring opening of R-D-glucose limits the rate, and on the alkaline side, ring closure of the open-chain sugar limits the rate. A mutarotation mechanism is presented in which one of the catalytic groups shuttles a proton to and from the endocyclic oxygen and the other two shuttle protons to the anomeric oxygen atoms. In this mechanism, three catalytic groups overcome the problem of nonstereospecificity in mutarotation. The groups are postulated to be His 104, His 175, and Glu 309. Mutations of these residues grossly impair catalytic activity. Variants H104Q-and E309Q-GalM display sufficient activity to allow profiles of log k cat /K m versus pH to be constructed. Both profiles show breaks on the acid side corresponding to pK a values of 5.8 for H104Q and 6.3 for E309Q. Apparently, ring opening of R-D-glucose limits the rate at low pHs, but ring closure does not become rate limiting at pHs up to 8.5 in reactions of these variants.
Biochemistry, 1992
The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the 0 2 and 0 4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 when it binds 0 1 of the substrate and by more than 1 A when it also binds 0 2 ; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions. DXylose isomerase (EC 5.3.1.5) catalyzes the isomerization of the five-carbon aldose D-XylOSe to the ketose D-xylulose. It also converts D-glucose, a six-carbon analog of D-xylose, to D-fructose in a reaction that is industrially applied to the production of high-fructose corn syrup. For this reason, Dxylose isomerase is widely known as glucose isomerase, even though the kinetic parameters are less favorable for the sixcarbon than for the five-carbon substrate. Aldose-ketose isomerization requires divalent metal cations such as MgZ+, Cd+, or MnZ+. Bacterial xylose isomerases have four identical subunits with molecular weights near 43 000. X-ray studies of the enzymes from several Streptomyces species have shown that they have very similar three-dimensional structures (
Journal of Chemical Education, 2011
b S Supporting Information B lood glucometers have been described for laboratory use several times in this Journal. 1À3 The most recent article showed that, at least in principle, a blood glucometer could be used to study the kinetics of the mutarotation of glucose. Because Rand β-D-glucose are optical isomers, a direct technique for monitoring the kinetic process of mutarotation in solution has been polarimetry, which requires a relatively expensive instrument. While studying the mutarotation of R-D-glucose, Perles and Volpe 3 found an excellent correlation between polarimeter readings and blood glucometer readings. However, the authors did not directly use a blood glucometer to obtain the kinetic parameters such as reaction order, reaction rate constant, and the Arrhenius activation energy.
The Oxidation of 1-Deuterated Glucose by Glucose Oxidase
Journal of Biological Chemistry, 1967
In order to trace the pathway of substrate-derived hydrogen in the mechanism of the glucose oxidase reaction, the kinetics of oxidation of D-glucose-l-2H was studied by conventional and stopped flow spectrophotometric methods.
The substrate specificity of immobilized D-glucose isomerase (EC 5.3.1.5) is investigated with an immobilized enzyme-packed reactor. A series of isomerization experiments with a-, band nd equilibrated D-glucose solutions indicates that b anomer as well as a anomer is a substrate of the glucose isomerase at pH 7.5 and 60°C. For substrate concentration of 0.028 mol l − 1 (1% w/v), the initial conversion rate of aD glucose was 43% higher than that with equilibrated glucose at the same concentration and 113% higher than b-D-glucose conversion rate. This anomeric reactivity of glucose isomerase is mathematically described with a set of kinetic equations based on the reaction steps complying with Briggs-Haldane mechanism and the experimentally determined kinetic constants. The proposed reaction mechanism includes the mutarotation and the isomerization reactions of a-and b-D-glucose with different rate constants.