156 ROBERT A. ALBERTY 



H 



X 'X 



+ H,0 =^=^ 



2 



C. ' X 



OC ^H -Qfi^ Vh 



OH 



Water is added to the double bond of fumarate to form the /-isomer of malate. 

 At equilibrium there is about four times as much /-malate as fumarate so that 

 the kinetics of both the forward and the reverse reactions may be studied. This 

 is of interest in checking certain conclusions as may be seen later. 



The catalysis is stereospecific at the hydroxyl carbon, and experiments by 

 Dr. Fisher (Fisher, Frieden, INIcKee and Alberty, 1955) using heavy water show 

 that it is stereospecific in addition at the methylene carbon atom. When the 

 reaction is carried out in deuterium oxide a single atom of deuterium is intro- 

 duced into /-malate, and when this monodeutero-/-malate is dehydrated enzy- 

 matically, the deuterium is lost and no deuterium is incorporated into the 

 fumarate. 



Fumarase crystallized (Massey, 1951; Frieden, Bock and Alberty, 1954) 

 from pig heart muscle has a molecular weight of 220,000 and appears to be a 

 simple protein. Fumarase has a turnover number of the order of 10* min.~^ 

 which depends upon the pH and buffer. 



A number of compounds have been tested as possible substrates for fumarase, 

 and none have been found to react. The list (JMassey, 1953) includes ^/-malate, 

 <//-thiomalate, maleate, cis and /ra»5-aconitate, citrate, mesaconate (which is 

 the methyl substituted fumarate), citraconate (which is the methyl substi- 

 tuted maleate), J,/and we^o-tartrate, Z-ethane-l-hydroxy-l-carboxylic-2-sul- 

 phonic acid and mono- and diesters of fumarate. Thus fumarase has a very high 

 degree of substrate specificity. A number of other compounds of interest remain 

 to be tested when they have been freed of fumarate and malate to a sutificient 

 extent. 



Dependence of Kinetics upon pH 



The kinetics of the fumarase reaction are markedly dependent upon pH 

 (Massey, 1953; Alberty, Massey, Frieden and Fuhlbrigge, 1954; Frieden and 

 Alberty, 1955) and the composition of the buffer. The anions in the buffer are 

 apparently responsible for the buffer effects which are both of an activating 

 and inhibiting nature. Phosphate buffers present an additional complication in 

 that as we go from low pH values to high pH values we go from a buffer which 

 contains primarily monovalent phosphate ions to one that contains primarily 

 divalent phosphate ions. Since these ions have quite different activating and 

 inhibiting effects on fumarase this complicates the elucidation of the ionization 



