11] STRUCTURE AND ACTIVITY OF PAPAIN 185 



The linear relationship between proteolytic activity and reactive thiol 

 shown in Fig. 1 provides strong evidence that the thiol group is directly 

 involved in the catalytic activity of the enzyme. It may be noted that the 

 thiol group of papain reacts very rapidly with PCMB. Indeed, the reaction 

 is essentially complete in less than two minutes. 



SH~MOLES/MOLE PROTEIN 



Fig. 1 . Proteolytic coefficient (Cj) for hydrolysis of benzoyl-L-argininamide as a func- 

 tion of sulfhydryl reactive with /7-chloromercuribenzoate for different preparations of 

 column-reduced crystalline papain. (Finkle and Smith, unpublished.) 



Less extensive studies of the reaction of iodoacetamide with the enzyme 

 also demonstrate that less than one equivalent of this substance is bound 

 by the enzyme.^ Moreover, this reaction produces complete and irreversible 

 inhibition of the enzyme. Reaction of the alkylating agent with a cysteine 

 residue should produce a thiol ether derivative (Fig. 2). Identification of this 



NoOH 

 ■SH-HICHgCONHg >- 



Nol 



-S-CH2CONH2 



HOOC-CHCH-S-CH,COOH 

 (I) 



NH- 



Performic, LgQ u-M 

 acid ( 3 HCl 



*- HOOC — CHCHoSOtH 



Fig. 2. Reaction of iodoacetamide with a sulfhydryl group and subsequent fate after 

 oxidation and hydrolysis to produce ^-carboxymethylcysteine sulfone (I). 



