Studies on the active--SH group of papain and on the mechanism of papain activation by thiols.

It is now generally agreed that papain possesses a single reactive sulfhydryl group which is essential for activity and which probably participates in the catalytic mechanism by forming acyl intermediates with the different substrates (2, 3). Smith (4) has claimed that the essential sulfhydryl group of papain is kinetically more active than most protein sulfhydryl groups and that it possesses different chemical properties. On the basis of indirect evidence, it was suggested that this sulfhydryl group is present in the form of an internal thioester (4), and the reaction mechanism recently advocated by Smith (4) was based on this concept. The study of the reactivity and chemical properties of the active -SH group of crystalline papain has so far been complicated by the fact that the preparations obtained were active only after addition of reducing substances (5) or when subjected to reducing procedures (6, 7). In this investigation, crystalline papain was obtained which was active without any special pretreatment or additions. This fact made it possible to study, in the absence cf interfering substances, the reactivity of the active -SH group of papain towards different types of sulfhydryl reagents. Also, the mechanism of papain activation, which is not clearly understood (3), has been investigated. The quantitative data presented here confirm the view that the -SH group of papain is highly reactive. However, they fail to support the hypothesis that it is present in the form of an internal thioester. The kinetic studies indicate that thiols may activate papain in part by increasing the rate of decomposition of the enzyme-substrate complex.