Thermostability of yeast hexokinase and yeast glucose-6-phosphate dehydrogenase

Kinetic study of the mechanism of the temperature-induced loss of the catalytic activity by yeast hexokinase (HK) and yeast glucose-6phosphate dehydrogenase (G-6-PDG) has shown the dissociative nature of the processes. In the temperature range 40–47°C, they are satisfactorily described in terms of consecutive reactions in which steps of irreversible denaturation of the monomeric units follow the reversible dissociation of inactive oligomeric forms into the active units, resulting in an increase in catalytic activity. The experimental data have been analyzed in the framework of the dissociative mechanism, and a semiquantitative method has been developed for calculating the individual rate constants.     

Overexpression of glucose-6-phosphate dehydrogenase in genetically modified Saccharomyces cerevisiae

Glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) is an abun dant enzyme in Saccharomyces cerevisiae. This enzyme is of great interest as an analytical reagent because it is used in a large number of quantitative assays. A strain of S. cerevisiae was genetically modified to improve G6PD production during aerobic culture. The modifications are based on cloning the G6PD sequence under the control of promoters that are upregulated by the carbon source used for yeast growth. The results showed that S. cerevisiae acquired from a commercial source and the same strain produced by aerobic cultivation under controlled conditions provided very similar G6PD. However, G6PD production by genetically modified S. cerevisiae produced very high enzyme activity and showed to be the most effective procedure to obtain glucose-6-phosphate dehydrogenase. As a consequence, the cost of producing G6PD can be significantly reduced by using strains that contain levels of G6PD up to 14-fold higher than the level of G6PD found in commercially available strains.     

Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Recycling of NADP⁺ using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP⁺ was recycled more than 10-fold. We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation.

1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm.
2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration.
3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K _M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2).

o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity. Protection against heat inactivation was obtained for the two activities with NAD⁺, NADP⁺, NADH, and NADPH. All these results suggested the possibility of recycling of NADP⁺ under similar conditions to those previously described for NAD⁺ (Burstein et al., 1981). It becomes thus possible to use various NAD⁺ and NADP⁺-dependent dehydrogenases in enzyme technology.     

Estimation of the biological variation of glucose-6-phosphate dehydrogenase in dried blood spots

Models based on biological variation provide a well-accepted database with reliable information for clinical laboratories for all purposes including screening, diagnosis and follow-up. Newborn screening laboratories use a blood spotted paper matrix to measure the analytes. This matrix medium is certainly different than body fluid matrix medium. After long-term monitoring of the performance of the Glucose-6-Phosphate Dehydrogenase Kit (R&D Diagnostics OSMMR 2000-D G6PD), the results obtained from the variation analysis were statistically evaluated. Analytical coefficient of variation, CV _A, was found to be 5.41%. The CV _A derived from between run assays was 5.32%, while within-subject biological coefficient of variation, CV _I, was 7.26%. Since minimum performance is defined as CV _A< 0.750 CV _I , CV _A should be lower than 5.44%. The analytical bias in calculation of total error was chosen to evaluate the performance of this assay. In this aspect, CV _G, between-subject biological coefficient of variation, was found to be equal to 10.35%. B _A was found to be 4.12%, which is lower than 4.74%, which means that it is acceptable. Therefore, the minimum quality specification for total error allowable is . When the relevant results obtained in this study were substituted in this formula, TE _a was found to be 13.7% for G6PD measurement in dried blood spots on paper filter matrix. It is expected that this figure will be helpful for the performance evaluation of newborn screening laboratories performing G6PD screening. We have been using error grid graphs for the evaluation of our external quality assurance survey results for the last two years, only because there was no data for assays employing filter matrix. Even the TE _a already reported for EDTA whole blood samples used in G6PD assays has been remarkably high, which can easily create the wrong impression that G6PD is not a reliable test to perform from blood spot cards. The present study shows that this assay is adequately reliable even when performed from dried blood spot matrix. However, we believe that the combination of total allowable error, error grid graphs with a well-defined cut off is the best approach to obtain an accurate performance evaluation for this test.Presented at the 10th Conference Quality in the Spotlight, March 2005, Antwerp, Belgium.     

Immobilized glucose-6-phosphate dehydrogenase as a substrate for solubilized epidermal growth factor receptor tyrosine kinase

Based on our previously reported solution assay protocol, a solid-phase assay for the tyrosine kinase activity of the epidermal growth factor receptor has been developed. Glucose-6-phosphate dehydrogenase, immobilized noncovalently on microtiter plates, was used as the substrate in the solid-phase assay. Phosphorylation of the immobilized substrate takes place in the presence of ATP and a solubilized epidermal growth factor receptor preparation. After washing off the soluble reaction mixture, the phosphotyrosine-containing dehydrogenase produced on the well surface is quantitated by an ELISA method using a polyclonal antiphosphotyrosine antibody, a second antibody conjugated with horseradish peroxidase, and finally theo-phenylenediamine reaction. The absorbance at 492 nm developed in the wells is a measure of the kinase activity of the solubilized receptor preparation. Putative inhibitors of receptor kinase can be conveniently incorporated in this assay system to test for potential inhibitory activity. This assay, being rapid and convenient, is useful in drug screening programs where a high through-put rate is required.     

Use of the thermostable glucose-6-phosphate dehydrogenase as a label enzyme in steroid enzyme immunoassays.

A thermostable glucose-6-phosphate dehydrogenase was assessed as a label enzyme in a testosterone enzyme immunoassay system. Enzyme labeling of a steroid with the enzyme was carried out by the N-succinimidyl ester method. A dose-response curve with a satisfactory sensitivity could be obtained by the use of the enzyme-labeled antigen. This enzyme should be useful in hapten enzyme immunoassays because of its relatively high stability.     

Specific detection of nicotinamide coenzymes by liquid chromatography and amperometric detection with immobilized glucose-6-phosphate dehydrogenase

A liquid chromatographic system for the specific and simultaneous detection of nicotinamide coenzymes is constructed by combining an immobilized glucose-6-phosphate dehydrogenase reactor with an amperometric system based on a phenazine methosulphate-mediated reaction, after separation on a reversed-phase column. The calibration graphs are linear from 0.05 to 20 nmol for all four coenzymes. The detection limits are 3.2, 5.2, 7.9 and 9.4 pmol for NADP⁺, NADPH, NAD⁺ and NADH, respectively. The enzyme reactor retains most of its original activity after repeated use for 2 months.     

The effect of poly(ethylene glycol) on the activity and structure of glucose-6-phosphate dehydrogenase in solution

The effect of poly(ethylene glycol), PEG, on the enzymatic activity of glucose-6-phosphate dehydrogenase (G-6-PDH) in the oxidation of glucose-6-phosphate (G-6-P), using NADP+ as co-enzyme was investigated. The enzymatic activity was determined by means of spectrophotometry in three different media: pure Tris–HCl buffer, solution of PEG400 (20 wt.%) and of PEG4000 (20 wt.%), both in buffer. Comparing the enzymatic activity values measured in pure buffer with those in the polymer solutions, an increase in the enzymatic activity of 20% was observed in the presence of PEG400 as well as in PEG4000. Calorimetric studies indicated the absence of preferential interactions between G-6-PDH and PEG400 or PEG4000. Nevertheless, the interaction enthalpy, ΔH_int, between NADP+ and PEG400 and PEG4000 amounted to −9.3 and −26.7 kJ/mol, respectively. Small angle X-ray scattering (SAXS) measurements were performed in a higher concentration range. Data analysis performed from SAXS curves by means of the intra-particle distance distribution function p(r) and Guinier plots yielded for G-6-PDH in pure buffer and PEG400 solutions radius of gyration, R_g, of about 70 Å and in PEG4000 solutions, R_g of about 40 Å. The latter has the same dimension as that found in the dimeric crystallographic structure of G-6-PDH, evidencing that G-6-PDH preserves its dimeric configuration in PEG4000 solution. On the contrary, different aggregates of G-6-PDH are formed in the presence of either buffer or PEG400. These findings show that the presence of PEG in solution can exert an effect on the enzyme structure and activity.     

Purification of glucose-6-phosphate dehydrogenase from baker’s yeast in aqueous two-phase systems with free triazine dyes as affinity ligands

To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.     

Radiation target analyses of free and immobilized glucose 6-phosphate dehydrogenase

The sensitivity of the enzyme glucose 6-phosphate dehydrogenase to ionizing radiation was examined under several conditions, including the presence of several free-radical scavengers. The enzyme was also irradiated when covalently bound to polyacrylamide beads whose structure is very similar to the polypeptide backbone of proteins. All the enzyme forms were irradiated in the frozen state with high-energy electrons from a linear accelerator. Surviving enzyme activity and surviving monomers were determined; the data were analyzed by target theory.Free-radical scavengers reduced the radiation target size of both the activity and monomers of the free enzyme, but not that of the immobilized enzyme activity. The target size of the activity of the free enzyme was that of a dimer mass, but in the case of the immobilized enzyme it was equal to the smaller mass of the monomer.Free-radical scavengers reduce the target size by modifying radiation energy transfer. The target size of the polyacrylamide-bound enzyme activity was expected to be very large since the connection between polyacrylamide and protein is a peptide bond which permits transfer of radiation-deposited energy. Several explanations concerning energy transfer are suggested for this result.     

D-glyceraldehyde-3-phosphate dehydrogenase

Examination of the properties ofEscherichia coli and rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenases (GPDHs) modified by 2,3-butanedione has shown that both tetrameric enzymes are stabilized, on selective modification of arginine residues (probably Arg 231), in an asymmetric state with only two active centers capable of performing the dehydrogenase reaction. The functionally incompetent active centers can be alkylated by iodoacetate or iodoacetamide in the case ofE. coli enzyme, but are inaccessible for these reagents in the case of rabbit muscle D-GPDH. These results are consistent with the idea that the two homologous enzymes share common principles of the protein design, but differ somewhat in their active centers geometries. Modification of the arginine produces marked changes in the shape of the charge transfer complex spectrum in the region of 300–370 nm, suggestive of the alterations in the microenvironment of the nicotinamide ring of NAD⁺, although the coenzyme binding characteristics remain largely unaltered. On arginine modification, the enzyme becomes insensitive to the effect of AMP on the kinetic parameters of p-nitrophenyl acetate hydrolysis reaction.     

Enhancement of riboflavin-mediated photo-oxidation of glucose 6-phosphate dehydrogenase by urocanic acid

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen-mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.     

Microsomal electrodes for reduced nicotinamide adenine dinucleotide and its phosphate,glucose-6-phosphate and ascorbate

Rat liver microsomes have been immobilized in a membrane by gelatin entrapment. The resulting membranes can be attached to an oxygen electrode to provide a sensor for several compounds. NADPH and NADH are determined by utilizing the liver microsomal NADPH oxidase activity, which is, at least in part, due to the cytochrome P-450 system. The calibration graph for NADPH is linear up to 1 mM. A multi-enzyme system localized in liver microsomes allows the determination of 20–800 μM glucose-6-phosphate. The non-enzymatic lipid peroxidation in liver microsomes, which is induced by ascorbate, allows 0.5–2.5 mM ascorbate to be determined.     

Effect of pH on the stability of hexokinase and glucose 6-phosphate dehydrogenase

Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the stability of HK and G6PDH was evaluated in this work. Baker’s yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as μmol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4°C. It was observed that up to 4 h both enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.     

Purification of human red cell glucose 6-phosphate dehydrogenase by affinity chromatography.

Human glucose 6-phosphate dehydrogenase associated with NADPH was efficiently bound with agarose-bound NADP, whereas the enzyme associated with NADP was poorly bound with agarose-bound NADP. After the elimination of haemoglobin from haemolyzate by treatment with DEAE-cellulose, the enzyme was converted into the NADPH-bound form and was applied on an affinity column. The enzyme was specifically eluted from the column by NADP in the elution buffer. A homogeneous enzyme preparation was obtained in high yield.     

Evolutionary chemistry approach toward finding novel inhibitors of the type 2 diabetes target glucose-6-phosphate translocase

A genetic algorithm (GA), driven by experimentally determined biological activities as a feedback fitness function, was used to propose novel small molecules as inhibitors of glucose-6-phosphate translocase (G6PT) in iterative rounds of evolutionary optimization. A straightforward polymer-supported synthetic sequence was implemented to synthesize molecules proposed by the GA, and the biological activities of the compounds were determined by a microsomal assay. Additional compound design strategies were integrated, such as Tanimoto similarity-based selection of starting materials and transfer of favored structure elements into a new chemical scaffold to identify more active and selective inhibitors.     

Highly selective and sensitive detection of NADP coenzymes using co-immobilized glucose-6-phosphate dehydrogenase/diaphorase reactors as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system

Two enzyme reactors prepared by the co-immobilization of two different glucose-6-phosphate dehydrogenases (G6PDH; from Leuconstoc mescenteroides (LM) and yeast (Y) and diaphorase are employed to enhance the sensitivity of NAD(P) coenzymes as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system. The NAD(P) coenzymes are recycled enzymatically during passage through the reactor in the presence of sufficient glucose-6-phosphate and oxygen in the carrier solution to produce a large amount of hydrogen peroxide, which is detected chemiluminometrically in the subsequent flow line. The G6PDH(LM)/diaphorase co-immobilized reactor is not specific between the NAD and NADP coenzymes, but shows a six fold selectivity towards NADP coenzymes compared to NAD coenzymes; the amplification factors for NAD and NADP coenzymes are 60 and 380, respectively, at a flow rate of 0.3 ml min(-1). In contrast, the G6PDH(Y)/diaphorase co-immobilized reactor is specific for NADP coenzymes with an amplification factor of about 600 (at a flow rate of 0.3 ml min(-1)). The detection limit is 6 fmol for both NADP(+) and NADPH.     

alpha-fluorinated phosphonates as substrate mimics for glucose 6-phosphate dehydrogenase: the CHF stereochemistry matters

Reported is a systematic study of the "fitness" (in terms of kcat/Km) of a series of phosphonate mimics of glucose 6-phosphate (G6P) as unnatural substrates for G6P dehydrogenase from Leuconostoc mesenteroides. The four G6P analogues (9, 10, 15a, and 15b) differ only in the degree of fluorination at the "bridging" phosphonate carbon. All have been synthesized from benzyl 6-O-trifluoromethanesulfonyl-2,3,4-tri-O-benzyl beta-D-glucopyranoside (6). The phosphonates with bridging CH2 (9) and CF2 (10) groups are cleanly obtained by direct displacements with the appropriate LiX2CP(O)(OEt)2 reagents (X = H, F) in 15 min at -78 degrees C. For the (alpha-monofluoro)alkylphosphonates (15a/b), homologation of 6 is achieved via lithiodithiane-mediated triflate displacement, followed by aldehyde unmasking [CaCO3, Hg(ClO4)2, H2O]. Addition of diethyl phosphite anion produces diastereomeric, (alpha-hydroxy)phosphonates 13a/b (1.4:1 ratio) which may be readily separated by chromatography. The stereochemistry of the minor diastereomer was established as 7(S) via X-ray crystallographic structure determination of its p-bromobenzoate derivative, 16b. Treatment of the major 7(R) diastereomer with DAST produces alpha-fluorinated phosphonate 14a, in modest yield, with inversion of configuration, as established, again, by X-ray crystallography. To our knowledge, this is first example of DAST-mediated fluorination of a (nonbenzylic, nonpropargylic) secondary (alpha-hydroxy)phosphonate and thus establishes the stereochemical course of this transformation. alpha-Deprotonation/kinetic quenching of 14a provides access to the 7(R)-epimer (14b). For all four protected phosphonates (7, 8, 14a, and 14b), diethyl phosphonate ester deprotection was carried out with TMSBr, followed by global hydrogenolytic debenzylation to produce the free phosphonates, as alpha/beta anomeric mixtures. Titrations of G6P itself and the free phosphonic acids provides second pKa values of 6.5 (1, bridging-O), 5.4 (10, bridging-CF2), 6.2 (14a, bridging-CHF), and 7.6 (9, bridging-CH2). Leuconostoc mesenteroides G6PDH-mediated oxidation and Lineweaver-Burk analysis yields normalized kcat/Km values of 0.043 (14b, bridging-7(R)-CHF), 0.11 (10, bridging-CF2), 0.23 (14b, bridging-CH2), and 0.46 (14a, bridging-7(S)-CHF) relative to G6P itself, largely reflecting differences in Km. The fact that kcat/Km increases by more than an order of magnitude in going from the 7(R)-alpha-monofluoroalkyl phosphonate (worst substrate) to the 7(S)-diastereomer (best substrate) is especially notable and is discussed in the context of the known phosphate binding pocket of this enzyme as revealed by X-ray crystallography (Adams, M. J. et al. Structure 1994, 2, 1073-1087).