INHIBITION OF ENZYMES 23 



The SH groups of the active center may belong to glutathione or some 

 related substance, bound in peptide linkage, and glutathione has been iso- 

 lated from purified 3-PGDH (Krimsky and Racker, 1952; Segal and Boyer, 

 1953; Racker, 1954 b). However, this has not been established and several 

 facts, such as the much greater reactivity of the enzyme SH groups to 

 iodoacetate, are not readily explained. Incubation of 3-PGDH with iodo- 

 acetate reduces the yield of glutathione obtained on tryptic digestion, as 

 expected if glutathione were involved in the inhibition, but no labeled 

 glutathione could be found following reaction with iodoacetate-C^^. Never- 

 theless, labeled acetylglutathione was isolated from 3-PHGD allowed to 

 react with labeled acetyl-P (Krimsky and Racker, 1954). Recent work has 

 proved that the three reactive SH groups belong to cysteine residues in the 

 polypeptide structure of the apodehydrogenase. Treatment of rabbit muscle 

 3-PGDH with iodoacetate- 1-C^* and subsequent digestion provided a pep- 

 tide of the following structure: 



NH, NH2 



Ileu-Val-Ser-Asp-Ala-Ser-Cys-Thr-Thr-Asp-Cys-Leu-Ala-Pro-Leu-Ala-Lys 



COO' 



and all the labeling occurred in this peptide (Harris et al., 1963). It is likely 

 that 3-PGDH contains three identical polypeptide chains, each associated 

 with an active site. The other cysteine SH group in the peptide above does 

 not react with iodoacetate unless the enzyme is denatured. The esterolytic 

 activity of 3-PGDH, with j9-nitrophenylacetate as the substrate, is readily 

 inhibited by iodoacetate (Taylor et al., 1963), so it was assumed that this 

 catalysis proceeds at or near the site at which 3-phosphoglyceraldehyde is 

 reacted, but that p-nitrophenylacetate acetylates the enzyme and eventually 

 inhibits. Pepsin digestion of 3-PGDH treated with p-nitrophenylacetate 

 yielded a peptide, Ala-Ser-CysSAc; this, like the work with iodoacetate, 

 indicates that glutathione is not involved in the inhibition (Cunningham 

 and Schepman, 1963). 



3-PGDH isolated from muscle is usually 80-90% in the oxidized state 

 and requires incubation with cysteine or glutathione for activation. In 

 the oxidized, or disulfide, state the enzyme does not react with iodoacetate, 

 and this may be responsible for some of the deviations in susceptibility 

 reported. For example, Chefurka (1954) found 3-PGDH from flies to be 

 more resistant to iodoacetate than the enzyme from rabbit muscle, no inhi- 

 bition being observed after 30 min at pH 7.1 with 0.09 mM iodoacetate, 

 but here it is difficult to determine the state of the enzyme during incu- 

 bation. If 3-PGDH is incubated with iodoacetate and then an activator 

 added, it appears that little inhibition occurs. 



