Mutant forms of beta-galactosidase with an altered requirement for magnesium ions.
By random approaches we have previously isolated many variants of Escherichia coli beta-galactosidase within a short contiguous tract near the N-terminus (residues 8-12 of wild-type enzyme), some of which have increased stability towards heat and denaturants. The activity of these mutants was originally analysed and quantitated in situ in activity gels without the addition of magnesium ions to the buffer system. We now show that the improved stability is only observable under such conditions of limiting magnesium ion concentrations or in the presence of appropriate concentrations of a metal chelator. In the presence of EDTA, purified preparations of one of these mutant enzymes were much more resistant to denaturants than wild-type, but this differential was completely nullified in the presence of 1 mM Mg2+. However, the stability of this mutant enzyme in EDTA was lower than that shown by it, or the wild-type enzyme, in the presence of magnesium ions. In addition, certain alterations within another N-terminal tract (residues 27-31 of wild-type) resulted in enzymes with greater dependence on Mg2+ than natural beta-galactosidase. We conclude that a small number of residue changes in a large protein can profoundly modulate the requirement for metal ion stabilization, allowing partial abrogation of this need in certain cases. Thus, some enzymes which require divalent metal ions for structural purposes only may be engineered towards metal independence.
Dunn
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CSIRO Division of Biomolecular Engineering, North Ryde, NSW, Australia.
Jennings
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D000595:Amino Acid Sequence; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D004492:Edetic Acid; D001616:beta-Galactosidase; D008274:Magnesium