Ribulose-5-phosphate isomerase

Figure 3. Plausible mechanism for the conversion of D-ribulose-5-phosphate isomerase to n-arabinose-5-phosphate by n-arabinose-5-phosphate isomerase. It is a least-motion model consistent with the mechanisms of similar enzymes (21-23).

Ribulose 5-phosphate is capable of a reversible isomerization to other pentose phosphates-xylulose 5-phosphate and ribose 5-phosphate. These reactions are catalyzed by two respective enzymes, viz., pentose-phosphate epimerase and pentose-phosphate isomerase, according to the scheme below ... 

The product of the PNP enzyme, FDRP 9 has been purified and characterised. The evidence suggests that FDRP 9 is then isomerised to 5-fluoro-5-deoxyribulose-1-phosphate 10, acted upon by an isomerase (Scheme 7). Such ribulose phosphates are well-known products of aldolases and a reverse aldol reaction will clearly generate fluoroacetaldehyde 11. Fluoroacetaldehyde 11 is then converted after oxidation to FAc 1. We have also shown that there is a pyridoxal phosphate (PLP)-dependent enzyme which converts fluoroacetaldehyde 11 and L-threonine 12 to 4-FT 2 and acetaldehyde in a transaldol reaction as shown in Scheme 8. Thus, all of the biosynthetic steps from fluoride ion to FAc 1 and 4-FT 2 can be rationalised as illustrated in Scheme 7. 

These enzymes vary widely in secondary and tertiary structure.1273 Mannose-6-phosphate isomerase is a 45 kDa Zn2+-containing monomer. The larger 65 kDa L-fucose isomerase, which also acts on D-arabinose, is a hexameric Mn2+-dependent enzyme.1273 L-Arabinose isomerase of E. coli, which interconverts arabinose and L-ribulose, is a hexamer of 60-kDa subunits128 while the D-xylose isomerase of Streptomyces is a tetramer of 43-kDa subunits.129 The nonenzymatic counterpart of the isomerization catalyzed by the enzyme is the base-catalyzed Lobry deBruyn-Alberda van Ekenstein transformation (Eq. 13-25).130... 

Alternatively, ribulose (D-eryt/iro-pentulose) 5-phosphate may be iso-merized to ribose 5-phosphate with pentose phosphate isomerase, but the same isomerase will convert D-ribose 5-phosphate into D- ryt/zro-pentulose 5-phosphate, the equilibrium being displaced by phosphorylation to the diphosphate (involving three enzyme systems). 

Ribulose-5-phosphate (3.13) can be converted to ribose-5-phosphate (3.14) and xylulose 5-phosphate (3.15), by the enzymes ribose-5-phosphate isomerase and ribulose 5-phosphate 3-epimerase, respectively. The two pentose-phosphate molecules, 3.14 and 3.15, are converted to a C3 and a C7 sugar-phosphate, glyceraldehyde 3-phosphate (3.4) and sedoheptulose-7-phosphate (3.16), respectively, via the action of atransketolase. 

D-Arabinose-5-phosphate isomerase, the first key enzyme in the synthesis of KDO, catalyzes the interconversion of D-ribulose-5-phosphate and D-arabinose-5-phosphate. This enzyme was briefly studied by Volk (18) and later by Lim and Cohen (19). Due to the instability of the enzyme, we have only purified this enzyme 100-fold. The reversible reaction is readily monitored by measuring the formation of the keto-sugar from the aldo-sugar by the method of Dische and Borenfreund (20). The K values for D-ribulose-5-phojsphate and D-arabinose-5-phosphate are 0.9 to 1.5 and 1 to 3 x 10 M, respectively. 

The 5-carbon sugar phosphates are interconverted by the action of epimerase and isomerase to yield ribulose-5-phosphate, which is phosphorylated by the enzyme ribulose phosphate kinase to make RuBP, the acceptor of C02. 

D-Ribulose 5-phosphate then undergoes an isomerization by ribose-5-phosphate isomerase to o-ribose 5-phosphate (Step 4) ... 

Figure 6.4. Role of transketolase in the pentose phosphate pathway. Glucose 6-phosphate dehydrogenase, EC 1.1.1.49 phosphogluconate dehydrogenase, EC 1.1.1.44 ribulose-phosphate epimerase, EC 5.1.3.1 phosphoribose isomerase, EC 5.3.1.6 transketolase, EC 2.2.1.1 and transaldolase, EC 2.2.1.2.

Ribose-5-phosphate isomerase, which catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate, activates the carbonyl differently. The aldose substrate can form a furanose (but not a pyranose) ring, but the furanose forms are so disfavoured (see Table 1.1) that there are likely to be appreciable amounts of open-chain sugar present at equilibrium. Moreover, this equilibrium, involving only furanose forms whose opening is possibly assisted by general acid catalysis from the 5-phosphate, is likely to be achieved rapidly. Although the enzyme from other sources crystallise with ring forms of the substrate bound, that from Thermus thermophilus crystallises with both ribose-... 

The isomerization step (reactions 13 and 14 in Table 22.1) involves the conversion of both ribose-5-phosphate and xylulose-5-phosphate to ribulose-5-phosphate. Ribose-5-phosphate isomerase catalyzes the conversion of ribose-5-phosphate to ribulose-5-phosphate, and xylulose-5-phosphate epimerase catalyzes the conversion of xylulose-5-phosphate to iibulose-5-phosphate (Figure 22.15). The reverse of both these reactions takes place in the pentose phosphate pathway, catalyzed by the same enzymes. 

Fig. 5. A simplified metabolic scheme of ethanol formation from glucose and xylose. Enzyme abbreviations GPDH Glucose 6-phosphate 1-dehydrogenase, PGDH Phosphogluconate dehydrogenase, PGI Glucose 6-phosphate-isomerase, RKI Ribose 5-phosphate isomerase, RPE Ribulose phosphate 3-epimerase, TAL Transaldolase, TKL Transketolase, XDH Xylitol dehydrogenase, XK-. Xylulokinase, XR Xylose reductase...

Now, finally, sedoheptulose 7-phosphate undergoes a transketolase-catalyzed (EC 2.2.1.2) process (as in Scheme 11.7) to remove two carbon atoms using the enzyme cofactor thiamine diphosphate to yield ribose 5-phosphate and a two-carbon fragment that has remained attached to the thiamine cofactor of transketo-lase (EC 2.2.1.1, sedoheptulose 7-phosphate) (Scheme 11.11). When the two-carbon fragment is added to glyceraldehyde 3-phosphate, the material of Scheme 11.8 again is applied and xylulose 5-phosphate results. The xylulose 5-phosphate isomerizes to ribulose 5-phosphate as in Scheme 11.9 (with intervention of ribulose phosphate 3-epimerase (EC 5.1.3.1). And, the ribose 5-phosphate, an aldose, isomerizes (an aldose-ketose isomerase, EC 5.3.1.6, ribose 5-phosphate isomerase) to ribulose 5-phosphate. 

Pentose Phosphate Isomerase. Ribulose-5-phosphate may undergo several reactions. One is isomerization to the corresponding aldose, ribose 5-phosphate (III). The widely distributed phosphopentose... 

In the studies described above, we had employed ribose 5-phosphate as the substrate, but the requirement for pentose phosphate isomerase in addition to transketolase suggested that the true substrate was ribulose 5-phosphate. However, this hypothesis was incompatible with the observations that sedoheptulose and fructose phosphates were substrates, since ribulose 5-phosphate possessed the opposite configuration at the critical C-3 position. The answer to this puzzle was provided by the discovery by Gilbert Ashwell and Jean Hickman at the National Institutes of Health of a new pentose ester, D-xylulose 5-phosphate, which Paul Stumpf in my laboratory also obtained as an intermediate in pentose fermentation in Lactobacillus plantarumP Jerard Hurwitz, then a post-doctoral... 

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. 

Most recently the enzyme of yeast catalyzing the cleavage of ribulose-5-phosphate has been crystallized and designated transketolase. " The pentose phosphate isomerase has been removed from the crystalline transketolase, which maintains its activity on ribulose-5-phosphate. The purified enzyme also catalyzes the formation of the ketopentose phosphate from glyceraldehyde-3-phosphate and a 2-carbon donor such as hy-droxypyruvate. Transketolase contains thiamine pyrophosphate as a coenzyme. 

CO2, present in the form of HCO3—, is fixed in the acceptor, ribulose-1,5-diphosphate, by means of the enzyme carboxydismutase. An intermediate with 6 C atoms is formed, the identity of which is still unknown. This substance is unstable. It decomposes into two molecules of 3-phosphoglyceric acid. The latter is then reduced to 3-phosphoglyceraldehyde by means of the ATP and NADPH + H+ formed in the primary processes. 3-Phosphoglyceraldehyde exists in equilibrium with its isomer, dihydroxy acetone phosphate. The equilibrium is controlled by the enzyme triose phosphate isomerase. 3-Phosphoglyceralde-hyde and dihydroxy acetone phosphate ar referred to collectively as triose phosphate. 

Fig. 36. The Calvin cycle (black lines) and pentose phosphate cycle (red lines). PGA = 3-Phosphoglyceric acid, PGAL = 3-phosphoglyceraldehyde, Rib. = ribose-5-phosphate, Xyl = xylulose-5-phosphate, Ru-diP = ribulose-1,5-diphosphate, C4 = erythrose-4-phosphate, FDP = fructose-1,6-diphosphate. A few of the enzymes participating are encoded, 1 = carboxydismutase, 2 = triose phosphate dehydrogenase, 3 = triose phosphate isomerase, 4 = aldolase, 5 = phosphatase, 6 = phosphoglucoisomerase. Details of the conversion of glucose-6-P into ribulose-5-P are given in Fig. 43. It should be pointed out that the pentose phosphate cycle presents only here and there a true reversal of the Calvin cycle. In many instances the mechanisms and enzymes are different.

The other two enzymes of this part of the photosynthetic carbon reduction cycle respond differently to illumination. Ribulose-phosphate kinase increases in activity by about 66%, but only after about 12 hours of illumination. Ribose-phosphate isomerase increases only by 50% after about 18 hours. These changes in activity do not occur in chloramphenicol-treated leaves. 

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