CIRCULAR DICHROISM STUDIES ON NATIVE AND PHENYLMETHANESULFONYL‐MESENTERICOPEPTIDASE

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.

pH DEPENDENCE OF THE CIRCULAR DICHROIC BANDS OF PHENYLMETHANESULFONYL‐MESENTERICOPEPTIDASE

“…The protein concentrations were deter-is dominated by large negative bands at 220 mined spectrophotometrically using a molar and 208nm, in agreement with the data reabsorption of 3.55 x lo4 M-' cm-' at 280nm ported previously (Genov & Shopova, 1978). (Genov, 1975).…”

“…The enzyme was inhibited with phenylmethanesulfonylfluoride (PMSF) to avoid the effect of autolysis. The introduction of a PMS-group in the active site of mesentericopeptidase does not change the CD spectra of the protein molecule (Genov & Shopova, 1978).…”

“…In a recent report (Genov & Shopova, 1978) we described circular dichroism (CD) studies on the denaturation of native and phenylmethanesulfonyl-mesentericopeptidase in the presence of urea. The enzyme molecule was unfolded in the presence of this reagent at neutral pH.…”