J. Biochem, 2003, Vol. 134, No. 4 599-606
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Mutagenesis of the Dimer Interface Region of Corynebacterium callunae Starch Phosphorylase Perturbs the Phosphate-Dependent Conformational Relay that Enhances Oligomeric Stability of the Enzyme
1 Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria; and 2 Institute of Biochemistry, Faculty of Sciences, Università Polytecnica delle Marche, Via Ranieri 60131 Ancona, Italy; and 3 Department of Chemical Sciences, University of Catania, 95125 Catania, Italy
We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in 2H2O solvent revealed small changes in residual amide II band intensities at 
1,550 cm–1, indicating that 1H/2H exchange in the wild type is clearly perturbed by the mutations. Decreased 1H/2H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45°C that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45°C and shows a higher 1H/2H exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40–50°C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4–5°C higher value.
* To whom correspondence should be addressed. Tel: +43-316-873-8400, Fax: +43-316-873-8434, E-mail: bernd.nidetzky{at}tugraz.at