REVERSIBLE DENATURATION OF MYOINOSITOL MONOPHOSPHATASE - THE STABILITY OF THE METAL-BINDING LOOP

The unfolding of bovine brain myo-inositol monophosphatase by guanidin e . HCl (Gdn . HCl) has been investigated. The recovery of circular di chroism, emission spectra, and catalytic activity after dilution of Gd n . HCl-treated samples indicate that the overall process is reversibl e. The steepness of the spectroscopic changes between 3 M and 5 M Gdn . HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process. The sensitized luminescence o f bound Tb(LII) was used as a probe of conformational changes of the m etal-binding loop. Denaturation of the enzyme by Gdn . HCl does not ab olish sensitized luminescence. A 50% decrease in sensitized luminescen ce was observed in 5 M Gdn . HCl. Under this set of experimental condi tions, the protein binds terbium with an association constant of 1x10( 6)M(-1). It is suggested that a residual structure of denatured inyo-i nositol monophosphatase is responsible for the binding of terbium ions . The kinetics of unfolding and refolding as a function of Gdn . HCl c oncentration were monitored by protein fluorescence in a stopped-flow instrument. The monophosphatase unfolded in a single kinetic phase wit h rate constants in the range 80-65 s(-1) at 25 degrees C. The refoldi ng kinetics fit monoexponential functions with rate constants in the r ange 120-65 s(-1) depending on the Gdn . HCl concentration. Substantia l refolding of the protein occurs within the dead time of mixing.