title: Role of Divalent Metal Ions on Activity and Stability of Thermostable Dipeptidase from Bacillus stearothermophilus.
abstract: Thermostable dipeptidase from Bacillus stearothermophilus, a typical metalloenzyme containing 1.0g atom of Zn per mole of subunit of the dimeric enzyme was markedly activated by exogenous divalent metal ions such as Mn(2+), Co(2+), and Cd(2+) . In contrast, several others including Ba(2+), Hg(2+), and Cu(2+) considerably inhibited the enzyme, even the inherent metal, Zn(2+), being slightly inhibitory. To study the metal-binding properties of this dipeptidase, the enzyme was completely resolved to the inactive, Zn-free apoenzyme by treatment with EDTA in the presence of guanidine hydrochloride in a weakly acidic buffer. The apoenzyme was readily reconstituted by incubation with either Zn(2+), Mn(2+), or Co(2+), restoring the catalytic activity. The Mn-reconstituted enzyme had nearly twice the activity of the original Zn-enzyme. Combined with kinetic analyses of reconstitution of the apoenzyme with metal ions, these results show that the enzyme has two non-identical metal-binding sites, each with a different property. Furthermore, substitution of Mn(2+) or Co(2+) for Zn(2+) considerably lowered the thermostability of the enzyme without affecting the overall conformation of the enzyme protein, suggesting that the prosthetic Zn is playing dual roles in conformational stability and catalysis of the thermostable dipeptidase.
authors.affiliation: a Department of Food Science and Biotechnology, Graduate School of Biotechnology , Korea University , Seoul 136-701 , Korea.
authors.forename: H Y
authors.initials: HY
authors.lastname: Cho
authors.affiliation: a Department of Food Science and Biotechnology, Graduate School of Biotechnology , Korea University , Seoul 136-701 , Korea.|b Division of Biological Science , Institute of Scientific and Industrial Research, Osaka University , Ibaraki, Osaka 567 , Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tanizawa
authors.affiliation: a Department of Food Science and Biotechnology, Graduate School of Biotechnology , Korea University , Seoul 136-701 , Korea.|c Department of Biotechnology, Faculty of Engineering , Kansai University , Suita, Osaka 564 , Japan.
authors.forename: K
authors.initials: K
authors.lastname: Soda
keywords: apoenzye; dipeptidase; metalloenzyme; reconstitution; thermostability