Tyr-503 of beta-galactosidase (Escherichia coli) plays an important role in degalactosylation

Substitutions for Tyr-503 of beta-galactosidase caused large decreases of t he activity. Both the galactosylation (k(2)) and degalactosylation (k(3)) r ates were decreased. Substitutions by residues without transferable protons , caused k(3) to decrease much more than k(2) while substitutions with resi dues having transferable protons, caused approximately equal decreases of k (2) and k(3). Several lines of evidence showed this. The K-m values of the substituted enzymes were much smaller than those for the wild type if the s ubstituted amino acid residues did not have transferable protons,this was n ot the case when the substituted residues had transferable protons. inhibit ion studies showed that the K-m values were not small because of small K-s values but were small because of relatively small k(3) values (compared wit h the k(2) values). The conclusion that the k(3) values are small relative to k(2) upon substitution with residues without transferable protons is als o based upon other studies: studies indicating:that the reaction rates were similar with different substrates, studies in the presence of alcohol acce pters, studies showing that the rate of inactivation by 2,4-dinitrophenyl-2 -deoxy-2-F-beta-D-galactopyranoside decreased much less than the rate of re activation; studies on burst kinetics, and pH studies. The data suggest tha t Tyr-503 may be important for the degalactosylation reaction because of it s ability to transfer protons and thereby facilitate cleavage of the transi ent covalent bond between galactose and Glu-537.