24 1. lODOACETATE AND lODOACETAMIDE 



It was first observed by Green et al. (1937 b) that the presence of a sub- 

 strate (in this case glyceraldehyde), can protect 3-PGDH against inactiva- 

 tion by iodoacetate, and this was confirmed for the pure enzyme and the 

 natural substrate. 3-P-glyceraldehyde, by Holzer and Holzer (1962), who 

 pointed out that the abihty of a substrate to protect is inversely propor- 

 tional to the K,„, as would be expected. Substrate protection has also been 

 recorded by Boyer and Segal (1953), Segal and Boyer (1953), and Krimsky 

 and Eacker (1954). The following do not protect against iodoacetate: NAD, 

 phosphate, and arsenate (which can replace phosphate in the transacyla- 

 tion). The failure of NAD to protect might be taken as evidence that the 

 NAD is not bound tightly to the SH group with which iodoacetate reacts, 

 or is not bound at this group at all. Indeed, Racker and Krimsky (1958) 

 found that the reaction with iodoacetate occurs more rapidly in the pres- 

 ence of NAD than in its absence, possibly indicating that NAD brings 

 about a structural change in the active center. Carboxymethylation of 

 approximately 5 SH groups of 3-PGDH is required for complete inactiva- 

 tion and, of these, 2 groups can be specifically protected by substrate (Boyer 

 and Segal, 1954). An SH group not involved in binding of NAD can be 

 protected by the substrate, since protection occurs in both the absence 

 and presence of bound NAD. Thus it may be that the combination of the 

 enzyme with the substrate does not involve an aldehydolysis of an S — NAD 

 bond but a direct reaction with another SH group. Since various phospho- 

 rylated substances which are not aldehydes (e.g. P-glycerate) can protect 

 the enzyme to some extent, the formation of a thiohemiacetal link is not 

 necessary, and it was postulated that a second point of attachment for 

 iodoacetate more related to the binding of phosphate may be assumed 

 (Krimsky and Racker, 1954). However, as pointed out many times pre- 

 viously, protection by a substance does not necessarily provide evidence of 

 the groups with which either the inhibitor or the protector reacts. 



A number of reactions are catalyzed by 3-PGDH under various conditions 

 and the effects of iodoacetate on these are often puzzling. The oxidation of 

 NADH by acetyl-P is well inhibited by iodoacetate, as expected, since this 

 is essentially the reverse of the normal reaction (Racker and Krimsky, 1952). 

 The oxidation of NADH by various dyes (a diaphorase-like action) is also 

 inhibited (Rafter and Colowick, 1957). However, the hydrolysis of acetyl-P 

 is not only not inhibited by 5 mM iodoacetate (Park and Koshland, 1958), 

 but may under certain conditions be accelerated by treatment with iodo- 

 acetate (Krimsky and Racker, 1955). This has been explained by assuming 

 that an alteration of the structure of the active center is required for this 

 hydrolytic activity, so that the usual acyl-enzyme complex is formed but 

 the reaction is diverted. Arsenolysis of acetyl-P is inhibited by iodoacetate 

 but not as readily as is the oxidation of 3-P-glyceraldehyde (Racker and 

 Krimsky, 1952; Kaplan et al., 1957); 1-2 mM iodoacetate may inhibit 25- 



