Ribulose preparation

Bromo-l,4-dihydroxybutan-2-one 1,4-bisphosphate (31) has been prepared from the protected bromohydrin of cw-but-2-ene-l,4-diol.95 Nucleophiles rapidly displace bromide ion from (31), and the latter has been used as an affinity label for ribulose-bisphosphate carboxylase. In this case two sites are labelled in each enzyme molecule, the two molecules of (31) being linked to the enzyme by two different lysine residues.96... 

The neutral FDPase and SDPase activities, which were present in the crude spinach extracts, were precipitated at lower ammonium sulfate concentration and could thus be separated from the specific alkaline FDPase. These activities appeared to be associated with the chloroplast fraction and did not require the presence of a divalent cation for activity. In crude extracts only the alkaline FDPase activity was inhibited by antiserum prepared by immunizing rabbits with the purified alkaline FDPase. The neutral FDPase was also active with ribulose diphosphate (RuDP) (98). 

A similar alkaline FDPase has also been obtained in highly purified form from Euglena gracilis (99) by heating and fractionation on DEAE-cellulose. The specific activity of the best preparation was approximately one-half that reported by Racker and Schroeder for the spinach enzyme. The enzyme appeared to be specific for fructose diphosphate, although SDP and ribulose diphosphate were not tested. The enzyme also required Mg2+ and was most active at pH 8.3 it showed very little activity at pH 7.5 or below. 

D-ribulose-L-cvsteine Prepared from D-ribose. M.p. 132-135°C (decomp.). 

Chloroplast fructose-1.6-bisphosphatase (FBPase) is one of the 13 enzymes of the Benson-Calvin cycle, all of them so far considered as solubles in the stromal space. However, the existence of some interactions with the thylakoid membranes has been reported in the last years concerning ribulose-1.5-bisphosphate carboxylase (1), FBPase (2,3), P-ribulokinase (4), and some other. In this context Kow and Gibbs (5) have obtained under mild conditions particulate preparations from spinach chloroplasts with low, but conclusive, CO assimilation capability without addition of any soluble stromal enzyme. On the other hand, loose association between some enzymes of the Benson-Calvin cycle has been also described (6). 

Biochemical analysis. Sample preparation and metabolite determinations were done essentially as described in (6). Ribulose-1,6-bisphosphate was determined by CO2-incorporation catalyzed by rubisco (7) and ATP by luciferase. Glucose 6-phosphate or carbohydrates were extracted and determined after (6) or (8), respectively. 

Ribulose-5-phosphate may undergo two different types of isomerization. The first involves carbon 1 and carbon 2 of the pentose to yield D-ribose-5-phosphate, and the second concerns carbon 3 and yields D-xylu-lose-5-phosphate. The first reaction is catalyzed by an isomerase, the second by an epimerase. Both enzymes are widely distributed in nature and have been found in most tissues purified isomerase was first prepared from alfalfa, and spleen epimerase has also been extensively purified. 

An extract has been prepared from spinach leaves which catalyzes the formation of D-ribulose diphosphate from adenosine triphosphate and D-ribose 5-phosphate (225). The D-ribulose diphosphate appears to be an intermediate in a carbon dioxide fixation system in which D-ribose 5-phosphate was the added substrate (226). 

The glycamines may serve as intermediates in the s3mthesis of 2-deoxy-sugars. 2-Deoxy-D-ribose was prepared by nitrous acid oxidation of 2-amino-2-deoxyribitol (SS8), obtained from D-arabinose and D-ribulose. 

Thiamine pyrophosphate has also recently been shown to exert a coenzyme function in the metabolism of pentose phosphate. - The formation of sedoheptulose phosphate from ribulose-5-phosphate by a highly purified enzyme preparation from spinach, which loses activity upon precipitation of the protein with ammonium sulfate at a low pH, was found to be almost completely reactivated by the addition of thiamine pyrophosphate. ... 

The mutual interconversion between pent-2-uloses and 2-C-(hydroxymethyl)-aldoses has been studied from the side of the former sugars only. The process has been exploited for the preparation of 2-C-(hydroxymethyl)-D-erythrose (35) from D-xylulose (34) (Scheme 14) and 2-C-(hydroxymethyl)-D-threose (37) from D-ribulose (36) [581 (Scheme 15). The thermodynamic equilibria of the respective interconverting pairs of sugars, 34 35 and 36 37, are 10 1 and... 

The rare sugar D-ribulose has been prepared from the chiral aldehyde (18) as shown in Scheme 2. An important feature of this synthesis is the first step in which 2-lithiofuran can be added with very high specificity (>95%) to (19) in the presence of zinc iodide, whereas in its absence little specificity is observed. It is therefore presumed that addition actually takes place to a zinc chelate of (18). 

Hexokinase catalysed syntheses of 6-amino-6-deoxy-D-glucose and -D-gluconate 6-phosphates, 6-amino-6-deoxy-D-fructose 6-phosphate and 1,6-bisphosphate, and 5-amino-5-deoxy-D-ribulose 5-phosphate have been reported. From a study of the properties and reactivity in enzymic reactions of these compounds, it was concluded that they were excellent isosteric analogues of the normal metabolic intermediates except for reactions catalysed by kinases. A preparative enzymic synthesis of uridine 5 -diphospho-2-acetamido-2-deoxy-D-glucose used chemically synthesized 2-acetamido-2-deoxy-D-glucose 1-phosphate and uridine triphosphate as reactants, and a calf liver enzyme extract containing uridine-5 -phospho-A-acetylglucosamine pyrophosphorylase. ... 

The discovery of ribulose-5-phosphate has posed the problem of whether this compound or the aldopentose phosphate is the immediate substrate for cleavage to triose phosphate. An enzyme preparation has been isolated from yeast by de la Haba and Racker, which will convert ribulose-5-phosphate to triose phosphate but which will produce this compound from ribose-5-phosphate only after a marked lag. The could be eliminated by another protein fraction from yeast presumably containing the pentose phosphate isomerase. However, it was observed that ribulose-5-phosphate alone is less readily cleaved to triose phosphate than is the reaction mixture of ribose-fi-phosphate and pentose isomerase. 

PGA Fomiation with Cell-Free Enj me Frepaiatioiis. The first successful attempt to obtain COt fixation in PGA was that of Fager (108), who found that spinach chloroplast preparations would form labeled PGA from C <3s provided they were supplemented with a heat-stable extract of spinach leaves or algae. A mixture of ribulose... 

Other reactions which are used for the preparation of some esters are those catal) ed by isomerases. Thus fructose 6-phosphate may be converted to glucose 6-phosphate, ribose 5-phosphate to ribulose 5-phosphate. Specific epimerases which lead to inversion at positions 3 or 4 have been employed. For instance D-xylulose 5-phosphate can be converted to L-ribulose 5-phosphate by a 4-epimerase, or to D-ribulose 5-phosphate by a 3-epimerase. 

D-Ribulose 5-phosphate has been prepared from 6-phosphogluconic acid with yeast and E. coh enzymes and separated by anion exchange . 

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