Beta-galactosidases (Escherichia coli) with double substitutions show that Tyr-503 acts independently of Glu-461 but cooperatively with Glu-537.

Beta-galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed. Control experiments showed that the very low kcat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins. Circular dichroism studies showed that the structures of the enzymes were intact. E461Q/Y503F-beta-galactosidase was inactivated in an "additive" manner. This indicated that Glu-461 and Tyr-503 act independently in catalysis. Because these residues are at opposite sides of the active site and act in different steps, this is expected. E537D/Y503F-beta-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent beta-galactosidases. This showed that Glu-537 and Tyr-503 interact cooperatively on the same step. This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose.