142 CARBON METABOLISM III 



later proved to have lost the capacity to form the acid from glucose 

 except under special circumstances (513, 550). Fumarate is also found 

 in the sporophores of basidiomycetes (575, 576). 



Penicillium resticulosum in glucose medium forms fumaryl-DL- 

 alanine, in which the amino group of alanine is joined in a peptide 

 linkage with one carboxyl of fumarate (68). 



Succinic acid accumulation is rarely so great as that of the other 

 acids considered thus far. Resting cells of Blastocladia pringsheimii 

 accumulate succinate equivalent to about 10 per cent of the glucose 

 metabolized (106), but the amounts formed by most organisms listed 

 in Table 1 are much smaller. Malic acid has only rarely (56, 465) 

 been reported to be formed in large amounts from glucose, although 

 it is produced by a variety of saprophytic fungi (Table 1). Most of 

 the identifications of malic acid, it should be noted, rest on color tests, 

 the use of which is often difficult and uncertain; re-examination by 

 enzymatic methods would be profitable. 



In fumaric acid synthesis, one of the most striking physiological 

 aspects is the effect of zinc, which increases growth and glucose con- 

 sumption and decreases, by as much as 80 per cent, the formation of 

 fumarate (208). Fumaric acid formation is thus a symptom of retarded 

 growth in the presence of an abundance of carbohydrate. By analogy 

 with acid formation in Aspergillus niger, it is likely that any metal 

 deficiency has the same effect (129, 130), but the role of other metals 

 is still uncertain and iron above its optimum level seems to have little 

 influence (208). 



Carbon dioxide has been shown by isotope studies to enter into the 

 molecule of fumaric acid (204, 205) and succinic acid (144). Succinate 

 formation by Streptomyces coelicolor and Blastocladia pringsheimii 

 is enhanced by carbon dioxide (106, 144), but the relation of exogenous 

 carbon dioxide to fumarate formation is uncertain (206, 207). 



The 4-carbon dicarboxylic acids under discussion probably are 

 formed by more than one biochemical mechanism. Four such mecha- 

 nisms have been suggested, namely: 



1. Oxidative formation in the citric acid cycle. Bulk formation by 

 this mechanism has not been proved. 



2. The reductive carboxylation of pyruvic acid, either to oxalace- 

 tate or directly, by the agency of the "malic enzyme," to malic acid. 

 This is the best known and perhaps the major pathway; evidence for 

 its role comes from studies on the incorporation of isotopic carbon 

 dioxide and on the physiological effects of carbon dioxide mentioned 

 above. 



3. The direct condensation of two 2-carbon fragments in the Thun- 



