274 2. ANALOGS OF ENZYME REACTION COMPONENTS 



1951; Saffran and Prado, 1949), and this is augmented by the addition of 

 cycle intermediates such as pyruvate or malate. These results indicate 

 clearly that aconitase is being inhibited intracellular ly. 



Essentially nothing is known of the possible effects of ^rans-aconitate on 

 cellular functions. No depression of Paramecium motility is seen at 10 mM 

 (Holland and Humphrey, 1953). However, the active transport of ions by 

 barley roots is markedly reduced (Ordin and Jacobson, 1955). K+ and Br" 

 uptakes are inhibited 32% and 33%, respectively, by 10 milf ^raws-aco- 

 nitate, and 63% and 47%, respectively, by 20 mM. These inhibitions are 

 probably not specific but the result of the depression of respiration. 



^rans- Aconitate is known to occur naturally in many plant tissues and 

 is abundant in sugar cane juice. It is formed from acetate-C^^ in corn tis- 

 sues and 95% of the aconitate which accumulates is in the trans form, it 

 being out of equilibrium with the cycle acids; further evidence for its com- 

 partmentalization during endogenous formation is provided by the fact 

 that it is metabolized quite readily when it is added to corn roots (Mac- 

 Lennan and Beevers, 1964). It was suggested by Rao and Altekar (1961) 

 that it may arise from ci'.s-aconitate through the mediation of an aconitate 

 isomerase, which they isolated from soil organisms. Some pseudomonads 

 are capable of metabolizing fraw^^-aconitate without previous exposure to 

 it and other strains can adapt to utilizing it (Altekar and Rao, 1963). 



FUMARASE 



Fumarase has been studied more intensively than most enzymes with 

 regard to interactions with competitive inhibitors, the effects of pH on 

 these interactions, and the nature of the active center. A generalized 

 representation of the bindings of fumarate and L-malate to the enzyme is 

 shown in Fig. 1-6-2, the pH effects are discussed in Chapter 1-14 (page 

 691), the apparent p^,'s for various competitive inhibitors are given in 

 Table 1-14-2, and the P-K'^'s of the two catalytically active sites for 

 fumarase and its substrate complexes are given in Table 1-14-3. 



Emphasis in this section will be directed to a more accurate delineation 

 of the active center configuration and to a more quantitative expression 

 of the ways in which competitive inhibitors interact with the active center. 

 Fumarase possesses four important groups: two cationic groups for binding 

 the COO" groups of the substrate in the trans position, and two ionizable 

 groups interacting with the groups on the a- and /5-carbon atoms and in- 

 volved in the addition or removal of water. The latter enzyme groups wiU 

 be designated as Rl and R^ in conformity with Wigler and Alberty (1960); 

 each may exist in the protonated form, RlH or Rj^H. The p^^'s of these 

 groups, which are 6.3 and 6.9 in the free enzyme, point to their phenolic 

 or imidazole nature; indeed, it is possible that these two groups are identical, 



