648 4. SULFHYDRYL REAGENTS 



rate of the enzyme reaction, particularly if it included the region of the 

 active center. 



(E) Tlie SH group reacted is on the substrate. This is a possibility especially 

 in the case of proteolytic enzymes, the modification of the peptide or protein 

 substrate preventing normal reaction with the enzyme. 



(F) The SH reagent interferes in a manner unrelated to SH groups. Many 

 SH reagents are not entirely specific for SH groups; e.g., iodoacetate also 

 reacts with amino groups and with heavy metal ions such as Cu++, and can 

 often form complexes with protein groups other than SH, especially amino 

 and carboxylate groups. Also the SH reagent may inhibit because it is 

 structurally similar to the substrate and can compete with it for the active 

 site; e.g., p-chloromercuribenzoate may act like a substituted benzoate on 

 certain enzymes rather than as a mercurial. 



Other mechanisms can be visualized in special cases and particularly for 

 those enzymes comprising several units and catalyzing complex reactions, 

 since the SH reagents can conceivably dissociate the functionally related 

 units, just as /j-chloromercuribenzoate can split the relatively simple muscle 

 phosphorylase a into four equivalent fractions (Madsen and Cori, 1956). 



It is very difficult to distinguish between the first four possibilities. In- 

 deed, proof of the functional role of SH groups usually must come from 

 evidence other than inhibition. Protection of the enzyme against SH re- 

 agents by the substrate does not provide adequate evidence that the react- 

 ed SH group is part of the active center, since the substrate could also slow 

 down or prevent reaction with vicinal groups as well, and could also sta- 

 bilize the protein structure around the active center. The secondary alter- 

 ation of protein structure brought about by reaction of SH groups cannot 

 always be detected by reversal experiments because the changes may, like 

 certain types of denaturation, be reversible. One must therefore conclude 

 that the demonstration of inhibition by SH reagents indicates at best (as- 

 suming the mechanisms (E) and (F) have been eliminated) only that one 

 or more SH groups are sufficiently near the active center to interfere with 

 the catalysis, either directly or by structural changes, when they are react- 

 ed. It must be admitted that such a conclusion is not very informative, 

 especially when it is considered that most enzymes contain 5 to 30 SH 

 groups per molecule and that statistically one would expect one or more 

 of these to be near the active center. Indeed, it is rather surprising that in 

 some instances a fair number of SH groups can be reacted without altering 

 the catalytic activity. 



One characteristic of inhibition by SH reagents which has been often 

 neglected is that the reaction with the enzyme SH groups in most cases 

 introduces a new side chain onto the protein. The inhibition may be as 

 much related, if not more, to the properties of this side chain as to the 



