BETA-GALACTOSIDASES OF ESCHERICHIA-COLI WITH SUBSTITUTIONS FOR GLU-461 CAN BE ACTIVATED BY NUCLEOPHILES AND CAN FORM BETA-D-GALACTOSYL ADDUCTS

Nucleophiles activated the catalytic actions of beta-galactosidases wi th neutral or positively charged substitutions for Glu-461. Aliphatic carboxylic acids increased the rate of hydrolysis of o-nitrophenyl bet a-D-galactopyranoside if the pKa values of the carboxyl groups were >s imilar to 3.5. Amino compounds activated if their pKa values were <sim ilar to 8.5. Imidazole, azide, and 2-mercaptoethanol also activated. N ucleophiles with high pKa values were able to activate the catalysis i f the pH was high, and this showed that the lack of activation at pH 7 .0 was because of protonation. Kinetic analysis showed that most of th e nucleophiles that activated were bound to the active site, since the activation followed Michaelis-Menten type saturation kinetics. The bi nding seemed to be dependent upon the hydrophobicity; the longer the a liphatic chain, the stronger the binding. Gas-liquid chromatographic a nalysis showed that adducts of some type were formed during the reacti ons in the presence of many of the nucleophiles. Three of these adduct s were purified and the nucleophiles were found beta-linked to D-galac tose. This indicates that if an intermediate covalent bond is formed i n the mechanism of beta-galactosidase action and if the nucleophile re acts to displace it, the intermediate covalent bond must have the alph a configuration and involve a group other than Glu-461.