4151-19-3

For baker's yeast transketolase (TK), cooperative binding of thiamine diphosphate (ThDP) and substrates in the transferase reaction is known. We show here that the differences in the properties of the active centers of TK are formed already upon the binding of Ca2+ in one of two initially identical subunits. When Ca2+ is bound in only one of the two active centers its affinity for the second decreases. The absence of a cation in the second active center decreases the affinity of ThDP to the first active center. Ca2+ binding increases the thermal stability of apo- and holoTK, i.e. changes the whole structure of the enzyme. Only in the presence of Ca2+, but not Mg2+, does the thermal stability of holoTK increase. In the one-substrate reaction in the presence of Ca2+, two Km are measured for the binding of xylulose-5-phosphate and hydroxypyruvate. For both substrates, Vmax of the first active center of holoTK, when it binds the substrate alone, is higher than of semiholoTK. When the substrate begins to bind also in the second active center, Vmax of both active centers decreases, which is explained by the previously shown flip-flop mechanism.

A chemical synthesis of ketopentose-5-phosphates that are involved in the pentose phosphate pathway has been developed. The ketopentose phosphates, d-ribulose-5-phosphate and d-xylulose-5-phosphate, were prepared in five steps starting from known intermed

An efficient and convenient platform for the facile synthesis of phosphorylated ketoses is described. All eight phosphorylated ketopentoses were produced using this platform starting from two common and inexpensive aldoses (d-xylose and l-arabinose) in more than 84% isolated yield (gram scale). In this method, reversible conversions (isomerization or epimerization) were accurately controlled toward the formation of desired ketose phosphates by targeted phosphorylation reactions catalyzed by substrate-specific kinases. The byproducts were selectively removed by silver nitrate precipitation avoiding the tedious and time-consuming separation of sugar phosphate from adenosine phosphates (ATP and ADP). Moreover, the described strategy can be expanded for the synthesis of other sugar phosphates.

The present invention is in the field of bioelectricity. The present invention provides energy generating systems, methods, and devices that are capable of converting chemical energy stored in sugars into useful electricity.

In this communication, we demonstrate that certain kinds of Rh porphyrins on carbon black can electrochemically oxidize aldose at low potentials. The onset potential was much lower than those with the other complex-based catalysts. A product analysis suggested that this reaction involves 2-electron oxidation of the aldehyde group.

D-Arabinitol 1-phosphate (Ara-ol1-P), a substrate for d-arabinitol- phosphate dehydrogenase (APDH), was chemically synthesized from d-arabinonic acid in five steps (O-acetylation, chlorination, reduction, phosphorylation, and de-O-acetylation). Ara-ol1-P was used as a substrate for the characterization of APDH from Bacillus halodurans. APDH converts Ara-ol1-P to xylulose 5-phosphate in the oxidative reaction; both NAD+ and NADP+ were accepted as co-factors. Kinetic parameters for the oxidative and reductive reactions are consistent with a ternary complex mechanism.

The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified. Copyright

Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribulose 5-phosphate catalyzed by a partially purified enzyme system for formaldehyde fixation in Methylomonas aminofaciens 77a.Much of the D-fructose 6-phosphate produced in this reaction was converted to D-glucose 6-phosphate by the addition of glucose-6-phosphate isomerase.A fed-batch reaction with periodic additions of the substrates afforded 56.2 g/liter D-glucose 6-phosphate and 26.8 g/liter D-fructose 6-phosphate.When methanol was used as the C1-donor, the yield of D-glucose 6-phosphate was high when alcohol oxidase was added.The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6 g/liter D-glucose 6-phosphate and 16.4 g/liter D-fructose 6-phosphate in 165 min.The molar yield of the total sugar phosphates to methanol added was 95percent.The addition of H2O2 and catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.

Tetrameric vanadate polyanion inhibits 6-phosphogluconate dehydrogenases from human, mammalian, yeast, and bacterial sources. The inhibition by a vanadate mixture containing monomer, dimer, and tetramer was determined by measuring the rates of 6-phosphogluconate oxidation and NADP (or NAD) reduction catalyzed by 6-phosphogluconate dehydrogenase. The inhibition by vanadate is competitive with respect to 6-phosphogluconate and mixed or noncompetitive with respect to NADP or NAD. 51V NMR spectroscopy was used to direcly correlate the inhibition of vanadate solutions to the vanadate tetramer. The measured inhibition constants with respect to 6-phosphogluconate for the tetramer are 0.078 mM for the human erythrocyte enzyme, 0.063 mM for the sheep liver enzyme, 0.013 mM for the yeast enzyme, and 0.24 mM for the Leuconostoc mesenteroides. The observed inhibition of 6-phosphogluconate dehydrogenase by vanadate tetramer is the first enzymatic activity observed of this polyanion. Our observations suggest the vanadate tetramer will be a potent inhibitor to other organic phosphate converting enzymes and preliminary results confirm this expectation. The vanadate tetramer may be an important species when considering the mechanism by which vanadium acts in biological systems in vitro and in vivo.

A synthetic strategy is developed that uses organic phosphate utilizing enzymes as catalysts and a mixture of an organic alcohol and inorganic arsenate or vanadate to replace the organic phosphate substrate.In this process, inorganic arsenate or vanadate reacts with the alcohol reversibly in situ to form a mixture of esters, one of which is accepted by the enzyme as a substrate.Examples of the utility of this approach are demonstrated in enzymatic aldol condensations catalyzed by fructose-1,6-diphosphate aldolase, fuculose-1-phosphate aldolase, and rhamnulose-1-phosphate aldolase with a mixture of dihydroxyacetone and inorganic arsenate as substrate.Several uncommon sugars and deoxy sugars are prepared on 5-17-mmol scales.Mechanistic studies on an aldol reaction indicate that the redox reaction between dihydroxyacetone and inorganic vanadate prohibits the use of such a mixture to replace dihydroxyacetone phosphate in enzymatic aldol condensations.

Substrate specificity of bakers' yeast transketolase has been studied: the enzyme appears to be enantioselective towards the acceptor aldehyde.L-erythrulose, D-xylulose and 5-deoxy-D-xylulose have been synthesized by spinach transketolase catalyzed condensation of hydroxypyruvate and the corresponding aldehyde.

This paper describes enzymatic syntheses of 5-phospho-D-ribosyl α-1-pyrophosphate (PRPP) on a 75-mmol scale.The reactions used PAN-immobilized PRPP synthetase as catalyst with in situ ATP-cofactor regeneration.In one procedure pure r-5-P was used as a starting material; in a second, r-5-P was synthesized by ribokinase-catalyzed phosphorilation of D-ribose and used in situ.The potential for use of PRPP as a starting material for the preparation of nucleotides was demonstrated by an enzymatic synthesis of UMP.This paper also describes several methods for the preparation of r-5-P: acid-catalyzed hydrolysis of AMP, acid-catalyzed hydrolysis of crude mononucleotide mixture obtained by digestion of RNA, chemical synthesis from D-ribose, and ribokinase-catalyzed synthesis from D-ribose.Procedures are described for the isolation of PRPP synthetase (from Salmonella typhimurium) and ribokinase (from Lactobacillus plantarum) and for immobilization of these enzymes in PAN.