Abstract
The conformational stability and reversibility of unfolding of the human dimeric enzyme Cu Zn superoxide dismutase (HSOD) and the three mutant enzymes constructed by replacement of Cys6 by Ala and Cys111 by Ser, singly and in combination, were determined by differential scanning calorimetry. The differential scanning calorimetry profile of wild-type HSOD consists of two components, which probably represent the unfolding of the oxidized and reduced forms of the enzyme, with denaturation temperatures (Tm) of 74.9 and 83.6 degrees C, approximately 7 degrees lower than those for bovine superoxide dismutase (BSOD). The conformational stabilities of the two components of the mutant HSOD's differ only slightly from those of the wild type (delta delta Gs of -0.2 to +0.8 kcal/mol of dimer), while replacement of the BSOD Cys6 by Ala is somewhat destabilizing (delta delta G of -0.7 to -1.3 kcal/mol of dimer). These small alterations in conformational stability do not correlate with the large increases in resistance to thermal inactivation following substitution of free Cys in both HSOD and BSOD (McRee, D.E., Redford, S.M., Getzoff, E.D., Lepock, J.R., Hallewell, R.A., and Tainer, J.A. (1990) J. Biol. Chem. 265, 14234-14241 and Hallewell, R.A., Imlay, K.C., Laria, I., Gallegos, C., Fong, N., Irvine, B., Getzoff, E.D., Tainer, J.A., Cubelli, D.E., Bielski, B.H.J., Olson, P., Mallenbach, G.T., and Cousens, L.S. (1991) Proteins Struct. Funct. Genet., submitted for publication). The reversibility of unfolding was determined by scanning part way through the profile, cooling, rescanning, and calculating the amount of protein irreversibly unfolded by the first scan. The order of reversibility at a constant level of unfolding is the same as the order of resistance to inactivation for both the HSOD and BSOD wild-type and mutant enzymes. Thus, the greater resistance to thermal inactivation of the superoxide dismutase enzymes with free Cys replaced by Ala or Ser is dominated by a greater resistance to irreversible unfolding and relatively unaffected by changes in conformational stability.
Highlights
From the Guelph- Waterloo Program for Graduate Work in Physics, Waterloo Campus and Department of Biology, University of Waterloo, Waterloo, Ontario NZL 3G1, Canada and the CChiron Corporation, Emeryville, California 94608
The conformational stability and reversibility of unfolding of the human dimeric enzyme Cu Zn superoxide dismutase (HSOD) and the three mutant enzymes constructed by replacement of Cys’ by Ala and Cys’” by
The transition temperatures are dependent on solvent composition and scan rate
Summary
The wild-type and mutant SODs (WT, C6S111, MCl11, A6S111) were constructed and isolated as previously described? Protein concentration was determined from the absorbance at 258 nm using an extinction coefficient of 10,300 M-l cm"". 2-5 were deconvoluted initially assuming irreversible unfolding and later assuming reversible unfolding to obtain values of t:..t:..G. For the irreversible case, the basic assumptions were that each peak represents an irreversible, one step transition of the form N -> D obeying pseudo-first order kinetics and that the temperature dependence of the rate constant for denaturation is approximated by the Arrhenius relation. During a DSC scan temperature increases uniformly with time: T(t)=To+vt where To is the initial temperature and v the scan rate Combining these equations gives: dtD[T(t») = eA-EA/RITo+ot) (1 - fn[T(t)j) dt which can be solved for tv as a function of temperature either numerically or by approximation as previously described (Lepock et al, 1990). The DSC profiles were deconvoluted assuming reversible denaturation using a modification of the algorithm proposed by Friere and Biltonen (1978) and Filimonov et al (1982)
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