Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.
To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-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 Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees 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 beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.
Harada
M
M
Institute for Biomedical Research, Suntory Ltd., Osaka, Japan.
Inohara
M
M
Nakao
M
M
Nakayama
T
T
Kakudo
A
A
Shibano
Y
Y
Amachi
T
T
D001665:Binding Sites; D004795:Enzyme Stability; D006358:Hot Temperature; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D016930:Saccharopolyspora / Q000201:enzymology*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism*
D008345:Manganese; D001616:beta-Galactosidase; D008274:Magnesium