Interaction of divalent cations with .beta.-galactosidase (Escherichia coli)

Abstract

Although the addition of various divalent metals to .beta.-galactosidase [EC 3.2.1.23] resulted in apparent activation, only Mg2+ and Mn2+ actually did activate. The apparent activation by the other divalent metals was due to Mg2+ impurities. Ca did not activate, but experiments suggested that it did bind. Other divalent metals which were studied failed to bind. The Kd for Mg2+ and Mn2+ were 2.8 .times. 10-7 and 1.1 .times. 10-8 M, respectively, and in each case 1 ion bound/monomer. These constants corresponded very closely to apparent values which were obtained from activation studies. The apparent binding constant for Ca2+, obtained from competition studies, was 1.5 .times. 10-5 M. Data were obtained which showed that Mg2+, Mn2+ and Ca2+ all compete for binding at a single site. Of interest and of possible molecular biological importance was the observation that, while Mg2+ bound noncooperatively (n = 1.0), Mn2+ did so in a highly cooperative manner (n = 3.4). The binding of Mn2+ (as compared to Mg2+) resulted in a 2-fold drop in the Vmax for the hydrolysis and transgalactosylic reactions of lactose but had little effect on the Vmax of hydrolysis of allolactose, p-nitrophenyl .beta.-D-galactopyranoside (PNPG), or o-nitrophenyl .beta.-D-galactopyranoside (ONPG); Km values were not affected differently for any of the substrates by Mn2+ as compared to Mg2+. When very low levels of divalent metal ions were present (0.01 M EDTA added) or when Ca2+ was bound with lactose as the substrate, a greater decrease was observed in the rate of the transgalactosylic reaction than in the rate of the hydrolytic reaction, and the Km values for lactose and ONPG were increased. Of the 3 divalent metal ions which bound to .beta.-galactosidase, only Mn2+ had significant stabilizing effects toward denaturing urea and heat conditions.