Fumaric acid labelled with

We have recently provided a stereospecific synthesis of samples of D-serine that are stereospecifically labeled at C-3 (94) (Scheme 23). This relies on the known (82) stereospecificity of the enzyme fumarase (EC 4.2.1.2) which adds water across the double bond of fumaric acid 72 with anti stereospecificity to give (2S)-malic acid 83. Thus [ H2]fumaric acid, 72, = H, in H2O... 

Homoserine also has been detected in filtrates of liver preparations incubated with methionine. Cantoni provisionally identified homoserine as a product of the acid hydrolysis of active methionine (S-adeno-sylmethionine). Beyond homoserine, the postulated reactions 2 and 3 are still more speculative. It might be presumed that homoserine is oxidized to aspartic acid, in analogy to the observations on the catabolism of lysine, in which the analogous a-amino adipic acid is an intermediate. If aspartic acid is formed, the subsequent reaction sequence is readily apparent. Evidence favorable to the proposed reaction pathway is the finding of Marshall and Friedberg, of the occurrence of a small amount of fumaric acid, labeled in the methine carbons, from the livers of mice injected with DL-methionine-2-C. ... 

Aspartic acid is readily converted to metabolites which are degraded to CO2. Stewart and Beevers (1%7) fed [U- C]aspartate to excised intact castor bean endosperms and found more than 50% of the added aspartate disappeared in the first hour with organic acids as the primary labeled products. Malate was the major organic acid labeled and the remaining label was almost entirely in fumarate. 

Several of the TCA cycle acids are so named because they accumulate to high concentrations in certain plant tissues— malic acid in apple Pyrus malm), citric acid in citrus fruits and fumaric acid in fumitory [Fumaria officinalis). Accumulations of this sort (there are a number of other examples) were initially taken as evidence that the TCA cycle was not operating rapidly in these plants. However when C-labelled TCA acids are fed to such tissues and then the specific activities (radioactivities per unit amount of carbon) of the evolved carbon dioxide and of the TCA acids within the tissue are measured it is found that whereas the specific activity of the carbon dioxide may be the same from two different acids, the specific activities of these acids in the cells may be very different indeed. This indicates that the whole cellular content of the acids does not participate in the TCA cycle, part of the acid is participating in the TCA cycle, part is remote from the site of this cycle. In certain crassulacean plants which contain high concentrations of malic acid it can be shown that there is a small metabolically active pool associated ivith the TCA cycle and a large pool which is relatively metabolically inert. Equilibration of label between these pools is slow and it is probable that the inert pool corresponds to the vacuole and the active pool with an intramitochondrial compartment. 

The pH dependence of the action of fumarate hydratase indicates participation of both an acidic and a basic group with pfCa values of 5.8 and 7.1.56 See Chapter 9 for additional information. However, either anion or carbocation mechanisms might be possible. That the cleavage of the C-H bond is not rate limiting is suggested by the observation that malate containing 2H in the pro-R position is dehydrated at the same rate as ordinary malate. If the anion mechanism (Eq. 13-13) is correct, the 2H from the pro-R position of specifically labeled malate might be removed rapidly, while the loss of OH could be slower. If so, the 2H would be "washed out" of L-malate faster than could happen by conversion to fumarate followed by rehydration to malate. In fact, the opposite was observed. 

In this connection it is of interest that Buchanan and coworkers demonstrated that isotopic acetoacetate (CH3 C 0-CH2 C 00H) and acetate (CH3-C OOH) are oxidized in kidney by way of the tricarboxylic cycle. In several experiments of this type, a-ketoglutarate, fumarate and succinate were isolated after incubation with isotopic acetoacetate or acetate. The isolated acids were found to contain sufficient excess of isotope to warrant the belief that acetoacetate and acetate are oxidized in kidney to a major extent by way of the tricarboxylic cycle. A similar result with carboxyl-labelled acetate has been obtained by Weinhouse and coworkers. ... 

As previously indicated, during dark fixation of CO2, no radioactive carbohydrate products are formed only organic and amino acids are significantly labeled. In cactus root tips, after a 2-h dark fixation period, 80% of the was in organic acids (Ting and Dugger, 1966) with a distribution of 66.4% in malate, 11.4% in citrate, and trace amounts in fumarate, succinate, and isocitrate. Because the tissue was actively respiring (about 200 pi g fr.wt h ), the data were taken to indicate compartmentation of the products outside the mitochondria. 

---