The role of sulfhydryl groups in the action and structure of mammalian α-amylase

Hog pancreatic α-amylase contains 2 -SH groups and 1 tightly bound Ca²⁺ per enzyme molecule. We have prepared derivatives of this enzyme by using 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), iodoacetamidonaphthol, and Hg²⁺. These derivatives retain enzymatic activity thereby demonstrating that the -SH groups of this enzyme are not required for enzymatic activity. The pH profile of these derivatives is the same as that of the native enzyme although the specific activity at each pH is depressed to a varying extent, depending on the derivative. The reaction with DTNB and iodoacetamidonaphthol proceeds only after prior removal of the tightly bound Ca²⁺. The Hg²⁺ derivatives, however, may be formed without prior removal of the Ca²⁺. Upon reacting the amylase with one mole of Hg²⁺ an -S-Hg-S- bridge is formed. This leads us to conclude that the 2 -SH groups lie very close to each other. These derivatives may be useful for an X-ray crystallographic study of this enzyme.