Beef heart malic dehydrogenases. VII. Reactivity of sulfhydryl groups and conformation of the supernatant enzyme.

Only 3 of the 6 sulfhydryl groups of native bovine heart supernatant malate dehydrogenase (S-MDH) react with p-mercuribenzoate (PMB) with no loss of enzymatic activity. In addition, prolonged incubation of S-MDH in 2.6 M urea solutions does not significantly alter the catalytic properties of the enzyme. In 2.6 M urea, however, all 6 sulfhydryl groups of the enzyme react with PMB, with attendant losses in catalytic activity. Under these conditions the rate of inactivation, although considerably slower than the rate of mercaptide formation, closely parallels the rate of change in the optical rotatory dispersion properties of the enzyme. Native S-MDH exhibits an optical rotatory dispersion curve with a distinct shoulder in the 280 to 290 rnp spectral region and a trough at 233 mp. Changes in these properties and other optical rotatory dispersion parameters were used to index conformational changes of S-MDH accompanying the various treatments. Native S-MDH and fully active SMDH . (PMB)3 exhibit identical optical rotatory dispersion curves. S-MDH in 2.6 M urea also exhibits an optical rotatory dispersion curve similar to that of the native enzyme. S-MDH . (PMB)3 and S-MDH . (PMB)G in 2.6 M urea, however, have significantly different optical rotatory dispersion properties, including a disappearance of the 280 rnp shoulder, decreases in [rr~‘]~~~ values, and large decreases in bo, uO, and X, values. Since the rate of change in these optical rotatory dispersion properties closely parallelled the rate of loss of catalytic activity but not the rate of mercaptide formation, the results were interpreted as indicating that the loss in activity is related to the changes in protein conformation rather than to the actual blocking of sulfhydryl groups.