Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions |
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Authors: | Dunn, I.S. Jennings, P.A. |
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Affiliation: | CSIRO Division of Biomolecular Engineering PO Box 184, North Ryde 2113 NSW, Australia |
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Abstract: | By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 812 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg2+. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 2731 of wild-type)resulted in enzymes with greater dependence on Mg2+ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence. |
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Keywords: | ß -galactosidase/ metalloproteins/ mutagenesis/ protein stabilization/ protein structure |
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