INHIBITION OF ENZYMES 295 



the latter case the molecule may be so oriented that the double bond is 

 directed away from the surface. It is likely that only those SH groups 

 can react when there are no charged groups in the immediate environment, 

 or when the pattern of charged groups allows maleate to assume the cor- 

 rect orientation. 



Let us summarize some of the possible ways in which maleate could in- 

 hibit enzymes. "(1) Reaction with SH groups. (2) Competition with anionic 

 substrates. These two mechanisms will be discussed in greater detail. (3) 

 Chelation of activating cations. Although maleate does not complex with the 

 common cations as readily as malonate, it is often used in such high concen- 

 trations that depletion of a metal cofactor could account for the inhibition. 

 The decarboxylating malate dehydrogenase of pigeon liver is inhibited by 

 maleate more potently when the Mn++ concentration is reduced, indicating 

 that at least part of the inhibition may be due to chelation of the Mn++ 

 (Stickland, 1959 b). (4) Chelation of enzyme-bound metal ions. Possibly 

 maleate can complex with metal cations which are associated tightly with 

 the enzyme active center, especially in view of the positions of the car- 

 boxylate groups, although no clear-cut example of this is known. It may 

 be that the potent inhibition of the oxidase activity of the copper-containing 

 ceruloplasmin by maleate is the result of metal ion complexing, since 

 fumarate and succinate are inactive (Curzon, 1960). However, the reaction 

 with SH groups cannot be eliminated, since Hg++ inhibits 50% at 0.01 mM. 

 (5) Reaction with substrate. The inhibition of the hydrolysis of ovalbumin 

 by pepsin observed with maleate was attributed by Crippa and Maffei 

 (1940) to reaction of the maleate with the ovalbumin rather than with 

 the pepsin. (6) Nonspecific ionic or ionic strength effects. When maleate is 

 used at higher concentrations (especially above 10 mM), one must consider 

 effects on the enzyme mediated through changes in the ionic strength 

 or by the cation accompanying the maleate. The inhibition of urease by 

 maleate and other anions at concentrations from 50 to 139 mM was inter- 

 preted in terms of an increase in ionic strength by Kistiakowsky et al. 

 (1952), with additional more specific contributions in certain cases. Native 

 aspartate transcarbamylase is stimulated by 0.5 mM maleate and inhibited 

 at higher concentrations; the native enzyme is a tetramer and possibly 

 maleate weakens this structure, but by what mechanism is unknown 

 (Gerhart and Pardee, 1964). 



Maleate is one of the least dependable detectors of enzyme SH groups. 

 However, it is just this property which might make it a useful inhibitor if 

 a certain enzyme or group of enzymes is attacked specifically. Although 

 maleate has not been tested on many enzymes, the available results point 

 to two important types of enzyme as being especially sensitive to maleate, 

 the a-keto acid oxidases and the transaminases, and certainly many of the 

 metabolic effects of maleate can be satisfactorily explained on the basis of 



