E461H-beta-galactosidase (Escherichia coli): altered divalent metal specificity and slow but reversible metal inactivation.
beta-galactosidase (Escherichia coli) with a His substituted for Glu-461 retained about 10% of its normal activity in the absence of divalent metals but was inactivated rather than activated by Mg2+, Mn2+, Zn2+, Ni2+, Cu2+, and Co2+. Since Zn2+, Ni2+, Cu2+, and Co2+ do not interact with wild type beta-galactosidase while Mg2+ and Mn2+ activate and Ca2+ binds but has no effect on wild type beta-galactosidase activity, the substituted enzyme has very different divalent metal interactions. A much larger amount of Mg2+ than of the other divalent metal ions was needed to inactivate the substituted enzyme at pH 7 (half-maximal activity was at 12.5 mM Mg2+ while the half-maximal activities with the other metals were at micromolar levels) compared to the amount of Mg2+ needed to activate the wild type enzyme. The inactivation of E461H-beta-galactosidase caused by Mg2+ took about 20 min. Reactivation by removal of the divalent metal took about 60 min. Interaction with Mg2+ was about 10(7)-fold stronger at pH 9 than at pH 7, and inactivation occurred in less than 2 min at higher pH values. "Galactosylation" (k2, cleavage of the glycosidic bond) seemed to be rate-limiting for E461H-beta-galactosidase at pH values above 6 with both o-nitrophenyl beta-D-galactopyranoside and p-nitrophenyl beta-D-galactopyranoside in both the presence and absence of Mg2+. Mg2+ caused decreases (about 50-fold) of the k2 values of E461H-beta-galactosidase (apparent pKa was about 6.8).(ABSTRACT TRUNCATED AT 250 WORDS)
Martinez-Bilbao
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M
Division of Biochemistry, Faculty of Science, University of Calgary, Alberta, Canada.
Gaunt
M T
MT
Huber
R E
RE
D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005690:Galactose / Q000378:metabolism; D018698:Glutamic Acid; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008961:Models, Structural; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D001616:beta-Galactosidase; D005690:Galactose