Purification and identification of the essential ionizable groups of honeybee, Apis mellifera L., trehalase

Abstract

Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl β-glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1% carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (Km) was 0.66 mM, and the molecular activity (k0) was 86.2s-1. The enzyme was an "inverting" type which produced β-glucose from α, α-trehalose. Dependence of the V and Km values on pH gave values for the ionization constants, pKe1 and pKe2, of essential ionizable groups 1 and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe1, and pKe2 were shifted to higher values of +0.2 and +0.5 pH unit, respectively. The ionization heat (ΔH) of ionizable group 1 was estimated to be +1.8 kcal/mol, and the ΔH value of group 2 was +1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (-COO-, ionizable group 1) and the other a protonated type (-COOH, ionizable group 2), although the pKe2 value is high.