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.     

Interaction of pyridoxal 5-phosphate with apo-serine hydroxymethyltransferase.

The interaction of pyridoxal 5-phosphate with beef liver serine hydroxymethyltransferase (5,10-methylenetetrahydrofolate:glycine hydroxymethyltransferase, EC 2.1.2.1) has been investigated using sedimentation velocity, kinetic and equilibrium techniques. No evidence for an aggregating system could be found in sedimentation velocity experiments in the presence or absence of pyridoxal 5-phosphate. Reassociation of pyridoxal 5-phosphate with apoenzyme and reacquisition of enzymic activity follow identical kinetics. An initial fast step is followed by a second order process with a rate constant of 66 M-1. s-1. A dissociation constant of 27.5 micrometer was obtained from equilibrium studies. No interaction of binding sites was exposed by altering pH or in the presence of glycine or folate. Maxima observed in pH profiles with both binding and reactivation are interpreted as the composite fo two overlapping processes, one of which is ionization of the pyridinium nitrogen of pyridoxal 5-phosphate and the other a functional group on the apoenzyme. Evidence is presented to indicate the necessity for the formation of an enzyme . pyridoxal 5-phosphate Schiff's base complex during catalytic turnover.     

Inhibition of ribose-5-phosphate isomerase by 4-phosphoerythronate.

Hoping to exploit the special affinity of enzymes for unstable intermediates in substrate transformation, we have determined the effectiveness of possible analogs of ene-diolate intermediates as inhibitors of spinach ribose-5-phosphate isomerase. 4-Phosphoerythronic acid was found to be a very strong competitive inhibitor, with a Ki value almost 3 orders of magnitude lower than the Km value of ribose 5-phosphate, and very much lower than the Ki value of any other inhibitor that was examined.     

Intact chloroplast electron flow. Effects of ribose 5-phosphate

Additions of ribose 5-phosphate to intact spinach chloroplasts were used to probe the effects of ADP regeneration on pH-gradient formation and electron-transfer reactions. In weakly illuminated chloroplasts, the ATP/ADP ratio dropped by 64% and the transthylakoid pH gradient decreased by a minimum of 0.2 units in response to ribose 5-phosphate. Nitrite reduction increased 2-fold while, under conditions of cyclic electron flow, the half-time for cytochrome f reduction decreased by a factor of two from 4.1 to 1.9 ms. The results suggest that metabolic ATP consumption, during the conversion of ribulose 5-phosphate to ribulose 1,5-bisphosphate, enhances electron transfer between plastohydroquinone and cytochrome f through decreases in the transthylakoid pH gradient caused by phosphorylation of ADP.     

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.     

Analogues of ribose 5-phosphate and 5-phosphoribosyl pyrophosphate. The preparation and properties of ribose 5-phosphorothioate and 5-phosphoribosyl 1-methylenediphosphonate

1. 5-Phosphoribosyl 1-methylenediphosphonate was isolated after reaction of ribose 5-phosphate and O-adenylyl methylenediphosphonate with 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. 2. The analogue reacted with adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase [K(m) (analogue)/K(m) (5-phosphoribosyl pyrophosphate) 0.17, 0.19 and 6.3 respectively; V(max.) (analogue)/V(max.) (5-phosphoribosyl pyrophosphate) 0.011, 0.26 and 1.1 respectively]. 3. The analogue was not a substrate for 5-phosphoribosyl pyrophosphate amidotransferase or orotate phosphoribosyltransferase. 4. Ribose 5-phosphorothioate was synthesized by allowing ribose to react with thiophosphoryl chloride in triethyl phosphate. The analogue was a substrate for 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. When this reaction was coupled to either adenine phosphoribosyltransferase or hypoxanthine phosphoribosyltransferase, adenosine 5'-phosphorothioate or inosine 5'-phosphorothioate was formed respectively.