KINETIC BEHAVIOUR OF SWEET ALMOND ALPHA-GALACTOSIDASE

The effect of temperature and pH on the activity of sweet almond α-galactosidase (α-galactoside galactohydrolase, EC 3.2.1.22) has been studied with melibiose and β-nitrophenyl α-d-galactoside (PNPG). Activation energies for the two substrates were found to be 12.4 and 19.0 kcal/mole, respectively. The pKm vs. t/T plots were linear for both substrates, and the enthalpies of binding were -7.3 and -10.2 kcal/mole for PNPG and melibiose, respectively. Changes in Km and vmax of melibiose with pH suggested the participation of a group with pK 5.75 in the enzymep-substrate binding and of another group with pK around 3 in the catalytic step. Similar studies with PNPG also revealed the presence of two groups (pK 3.4 or above and 6.7 or below), the pK values of which were strongly influenced by the substrate concentration. The group dissociating in the lower pH range appears to play a minor part only in the PNPG hydrolysis. In spite of apparently marked differences in the effect of temperature and pH, all the substrates appear to be hydrolysed at the same site on the enzyme molecule. The differences probably arose from differences in the mechanisms of the reaction or in the nature of the rate-limiting step in the two cases. © 1969.