OLIGOPEPTIDASE-B - CLONING AND PROBING STABILITY UNDER NONEQUILIBRIUMCONDITIONS

Oligopeptidase B is a member of a new serine peptidase family, unrelat ed to the trypsin and subtilisin families, It is a potential processin g enzyme of prokaryotes, being very specific for the basic amino acid pairs of polypeptides. An understanding of the kinetics of the enzyme requires the examination of its conformational stability under a varie ty of conditions, To this end, the enzyme was cloned from Escherichia coli HB101 by the PCR method, expressed with high yield in E. coli XL1 -Blue, and purified essentially in two chromatographic steps, The dena tured enzyme failed to refold, which precluded the calculation of free energy of stability, Delta G(0). Therefore, the unfolding rates were measured to probe the stability against urea, pH, and heat, Denaturati on processes were monitored by intrinsic fluorescence, circular dichro ism, and activity measurements, A static method, intrinsic fluorescenc e vs, pH, was indicative of significant changes in the tertiary struct ure of the enzyme pH < 6 and pH > 8.5, The more sensitive dynamic meth ods, unfolding rates in urea and inactivation rates at high temperatur e, revealed increased flexibility in the protein structure between pH 6 and pH 7, where the static method did not shaw significant changes, Inactivation of the enzyme in the acidic pH range correlated with the results obtained with the static rather than with the dynamic method, Acid denaturation at pH 3 was markedly retarded by 1 M NaCl, Against h eat. inactivation the enzyme was also considerably protected in the pr esence of salt, and the higher enthalpy and entropy of activation sugg ested the importance of hydration in the stabilization. The kinetics o f unfolding followed single-exponential decay under strongly denaturin g conditions (high urea concentration or high temperature), but deviat ed from the apparently two-state mechanism at low urea concentrations and at slightly acidic pH, The results indicate that under harsher den aturing conditions there is a single rate-limiting step in unfolding w hereas under milder conditions partly unfolded intermediates are popul ated. (C) 1998 Wiley-Liss, Inc.