The properties of β-galactosidases (Escherichia coli) with halogenated tyrosines

Abstract

An Escherichia coli tyrosine auxotroph (MR1) with an inducible lacZ was generated by mutagenesis. Of several tyrosine derivatives tested, only m-fluorotyrosine supported the growth of this mutant and allowed synthesis of active β-galactosidase. The pH profiles of the β-galactosidase that was obtained when this mutant was grown on m-fluorotyrosine (81.5% of the tyrosine was replaced by m-fluorotyrosine) indicated that a tyrosine may be acting as a general acid–base catalyst and that it (or another tyrosine with the same pKa) may be involved in substrate binding. Inactivation of normal β-galactosidase by treatment with lactoperoxidase in the presence of I− did not affect affinity-column binding, but incubation of this iodinated β-galactosidase with chymotrypsin caused a rapid degradation of a portion of the treated enzyme equal to the portion of the activity that was lost. A study with 125I− showed that the rapid degradation was mainly confined to iodinated molecules of enzyme. These studies indicate that iodination of β-galactosidase does not affect binding ability, but causes the enzyme to lose catalytic activity and become susceptible to chymotryptic action. Chloroperoxidase also caused rapid inactivation of normal β-galactosidase in the presence of Br− or I−, but there was a lag followed by a slow inactivation in the presence of CI−.Key words: β-galactosidase, tyrosine, halogenation, lactoperoxidase, mechanism.