Characterization of two trehalases in baker's yeast

Abstract

Trehalase activities at pH 5 (not inhibited by EDTA) and pH 7 (inhibited by EDTA) were present in the soluble fraction of disintegrated commercial baker''s yeast. The pH 5 activity binds strongly to concanavalin A, is only partially salted out by saturated (NH4)2SO4, has an apparent MW of 215,000 (by gel filtration) and is an acidic protein. It has a Km of 1.4 mM, a broad pH optimum (at 40 mM-trehalose) between pH 4 and 5, and is activated by .apprx. 30% by 20-300 mM neutral salts such as KCl, NaNO3 and MnCl2. The enzyme is strongly inhibited by acetic acid/acetate buffers, with a Ki of .apprx. 15 mM-acetic acid. The pH 7 activity does not bind to concanavalin A, is salted out at 20-32% (wt/vol) (NH4)2SO4 and has a MW of 170,000 (by gel filtration). It is absolutely dependent on Ca2+ or Mn2+ (Mg2+ is ineffective) and strongly inhibited by neutral salts in the 20-100 mM range. It can be activated by treatment with MgATP in the presence of cAMP. Activation decreases, but does not abolish, the Ca2+ requirement, and does not change the Km for trehalose (5.7 mM) or shift the sharp pH optimum at pH 6.7 (at 40 mM-trehalose).