REVERSIBLE UNFOLDING OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE - ACTIVE-SITE CAN BE RECONSTITUTED BY A NUMBER OF PATHWAYS

Acid-induced and guanidine hydrochloride (GdnCl)-induced reversible un folding of Escherichia coli alkaline phosphatase (AP) was characterize d under equilibrium conditions. The protein was exposed to extreme con ditions of pH 2.0 or 6 M GdnCl and was subsequently returned to normal conditions, Associated changes in the protein structure was probed by various spectroscopic methods, The changes in the functional properti es were monitored by measuring enzymatic activity, capacity to renatur e spontaneously upon removal of the denaturant, and renaturation in pr esence of various site-specific and nonspecific effector molecules, in the absence and presence of beta-mercaptoethanol. Analysis of the flu orescence and CD spectra showed that the unfolding of the organized st ructures was much more extensive in 6 M GdnCl than at pH 2.0. Intracha in S-S bonds in each unfolded state were accessible to reduction by be ta-mercaptoethanol. The effecters Zn2+ and ATP induced renaturation of active site only under reducing conditions, whereas Triton X-100 or a lpha-crystallin needed the presence of some organized structure. The r econstituted protein from each denatured state without or with an effe ctor showed different CD spectra. It is concluded that the active site domain of AP could be reconstituted independently of other structural domains in different pathways. (C) 1996 Academic Press, Inc.