Circular dichroism studies on native and phenylmethanesulfonyl-mesentericopeptidase. Denaturation in the presence of urea.

Abstract

At neutral pH the far ultraviolet circular dichroism (CD) spectrum of alkaline mesentericopeptidase is dominated by two negative bands: at 208 and 220 nm. The near ultraviolet CD spectrum is characterized by a large negative band at 277nm, a shoulder near 286nm and a small positive band at 297nm. The introduction of phenylmethanesulfonyl (PMS) group on the serine residue at the active site does not alter the intensity and the wavelength position of the bands.In contrast to the other alkaline bacterial proteases, which are remarkably resistant to denaturation by urea, mesentericopeptidase was unfolded in the presence of this reagent at neutral pH. The denaturation reactions in the presence of 9.5 M urea of both native and PMS‐mesentericopeptidase followed first order kinetics. The introduction of PMS‐group on the active‐site serine lowered the conformational stability of the enzyme. In the first 3h PMS‐mesentericopeptidase was denaturated 2.5 times faster than native protease.In the presence of 9.5 M urea at natural pH alkaline mesentericopeptidase lost its catalytic activity. The loss of enzymatic activity correlated with the unfolding of the protein molecule and also followed first‐order kinetics.