22 1. lODOACETATE AND lODOACETAMIDE 



The over-all reactions are the same in both schemes. There are two general 

 types of reaction involved, an oxidoreduction and a transacylation (from 

 the enzyme to phosphate). Since there is evidence that these can occur at 

 different loci on the enzyme, 3-PGDH has been called a double-headed 

 enzyme by Racker (1961), and we shall see that each reaction may be 

 studied alone. For this reason, it is likely that the schemes above do not 

 accurately represent the catalysis. It is actually not known if the same SH 

 group is involved in the binding of NAD, substrate, and iodoacetate and, 

 indeed, we shall discuss evidence that more than one SH group is functional 

 (protection experiments and failure of iodoacetate to inhibit certain reac- 

 tions catalyzed by 3-PGDH). 



Whatever the detailed mechanism of the reactions, it appears certain that 

 iodoacetate carboxymethylates one or more SH groups at or near the active 

 center and thereby interferes with the binding of NAD and substrate. 3- 

 PGDH is faintly yellow in concentrated solution and the color disappears 

 on reaction with iodoacetate; the change in absorption is best determined 

 at 360 m//, the approximate maximum of a broad absorption band charac- 

 teristic of the complex of the apodehydrogenase with NAD (Racker and 

 Krimsky, 1952). Incubation of the apodehydrogenase with iodoacetate also 

 prevents the appearance of the band upon addition of NAD. This has gen- 

 erally been taken to mean that NAD is bound through an SH group reac- 

 tive with iodoacetate, but does not necessarily follow since the steric inter- 

 ference of a carboxymethyl group might be exerted on the binding of NAD 

 as long as the SH group is in the vicinity of the site. However, thiols such 

 as glutathione complex with NAD nonenzymically, resulting in light ab- 

 sorption in a band centering around 335 m//. The reduction in the absorp- 

 tion at 360 m// brought about by iodoacetate is certainly parallel to the 

 loss of catalytic activity (see accompanying tabulation) (Racker, 1954 b; 



Iodoacetate 



, . , ^ > Optical density at 360 mfi (%) Activity (%) 



(equivalents) 



None 100 100 



1 66 69 



2 26 28 



3 



Racker and Krimsky, 1958). This reconstitued muscle 3-PGDH contained 

 3 moles of NAD per mole of enzyme. It is interesting that although iodo- 

 acetate reacts rapidly with the enzyme-NAD complex, iodoacetamide reacts 

 very sluggishly, indicating some importance of the COO" group and provid- 

 ing some basis for the occasional observations that iodoacetamide is not as 

 effective an inhibitor of glycolysis as iodoacetate. 



