Simple Procedures for the Preparation of d-Ribose-5-phosphate Ketol-Isomerase,d-Ribulose-5-phosphate 3-Epimerase and d-Sedoheptulose-7-phosphate: d-Glyceraldehyde-3-phosphate Glycolaldehydetransferase

d-Ribose-5-phophate ketol-isomerase (EC 5.3.1,6), d-ribuIose-5-phosphate 3-epimerase (EC 5.1.3.1) and d-sedoheptulose-7-phosphate: d-gIyceraldehyde-3-phosphate glycolaldehyde-transferase (EC 2.2.1,1) have been partially purified. d-Ribose-5-phosphate ketol-isomerase was purified from spinach by column chromatography with DEAE-cellulose and DEAE-Sephadex A-50; d-ribulose-5-phosphate 3-epimerase was purified from baker’s yeast by column chromatography with DEAE-cellulose; and d-sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase was purified from a Bacillus species No. 102 mutant G3–46–22–6 by column chromatography with DEAE-cellulose. The preparations were used for the determination of the activities of these enzymes in the parent and d-ribose-forming mutants of a Bacillus species.     

Arabinose 5-phosphate covalently inhibits transaldolase

Arabinose 5-phosphate (A5P) is the aldopentose version of the ketohexose fructose 6-phosphate (F6P), having identical stereochemistry but lacking atoms corresponding to the 1-carbon and 1-hydroxyl. Despite structural similarity and conservation of the reactive portion of F6P, F6P acts as a substrate whereas A5P is reported to be an inhibitor of transaldolase. To address the lack of A5P reactivity we determined a crystal structure of the Francisella tularensis transaldolase in complex with A5P. This structure reveals that like F6P, A5P forms a covalent Schiff base with active site Lys135. Unlike F6P, A5P binding fails to displace an ordered active site water molecule. Retaining this water necessitates conformational changes at the A5P-protein linkage that possibly hinder reactivity. The findings presented here show the basis of A5P inhibition and suggest an unusual mechanism of competitive, reversible-covalent transaldolase regulation.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

Characterization of enzymatic D-xylulose 5-phosphate synthesis

In this article we report on the characterization of the enzymatic synthesis of D-xylulose 5-phosphate using triosephosphate isomerase and transketolase. Two potential starting substrates are possible with this scheme. The data presented here allow a comparison of both routes for the synthesis, based on experimental information on reaction kinetics. Operational guidelines are proposed which should assist in the scale-up of such syntheses.     

Purification and characterization of ribitol-5-phosphate and xylitol-5-phosphate dehydrogenases from strains of Lactobacillus casei.

A simple three-step procedure is described which yields electrophoretically homogeneous preparations of ribitol-5-phosphate dehydrogenase and xylitol-5-phosphate dehydrogenase. The former enzyme is a 115,000-molecular-weight protein composed of two subunits of identical size and is specific for its substrate, ribitol. The xylitol-5-phosphate dehydrogenase exists as a tetrameric protein with a molecular weight of 180,000; this enzyme oxidizes the phosphate esters of both xylitol and D-arabitol. Characterization of the physical, kinetic, and immunological properties of the two enzymes suggests that the functionally similar enzymes may not be structurally related.     

Brain pyridoxine-5-phosphate oxidase. Modulation of its catalytic activity by reaction with pyridoxal 5-phosphate and analogs

Pyridoxine-5-P oxidase, the flavoprotein involved in the oxidation of pyridoxamine-5-P and pyridoxine-5-P to pyridoxal-5-P, has been isolated and purified to homogeneity using sheep brain tissues. Inactivation of the oxidase by bis-pyridoxal-5-P results in binding of the inhibitor to specific lysyl residues. After NaBH4 reduction of the inactivated enzyme, it was found that 1 P-pyridoxyl-pyridoxine-P residue was incorporated per enzyme dimer. After trypsin digestion of the bis-PLP modified enzyme, only one peptide absorbing at 320 nm, was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the labeled peptide was determined by automated Edman degradation. The observations reported in this paper are relevant to the mechanisms underlying the regulation of the catalytic function of pyridoxines-5-P oxidase by the product pyridoxal-5-P. It is postulated that the catalytic function of the oxidase is modulated by binding of pyridoxal-5-P to a specific lysyl residue of the dimeric structure of the protein.     

Kinetic properties of transketolase from the rat liver in a reaction with xylulose-5-phosphate and ribose-5-phosphate

An analysis of steady-state kinetics of purified rat liver transketolase shows that the reaction proceeds according to a two-stroke substitution ("ping-pong") mechanism. Based on the kinetic data, a competitive relationship was shown to exist between xylulose-5-phosphate and ribose-5-phosphate for the sites of substrate binding by the substituted form of the enzyme with the formation of a non-productive abortive complex (kd = 125 microM). The values of constants of two monomolecular steps of the reaction (k2 = 42 s-1; k4 = 9.4 s-1) were determined. It was assumed that the maximum rate-limiting step of the transketolase reaction is the degradation of the substituted form of transketolase--ribose-5-phosphate complex having a rate constant of k4.