To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable β-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca2+-specific sites (class II(Ca)) and one Mn2+-specific site (class II(Mn); K(d) for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (K(d), 1.2 μM) and Mg2+ (K(d), 2 μM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative V(max) (V(rel)), 100%) by a chelating resin at 4 °C yielded a less thermostable Ca1 species (V(rel), 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 °C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class II(Mn) site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class II(Ca) sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 °C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular β- galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology