Reversible Unfolding ofEscherichia coliAlkaline Phosphatase: Active Site Can Be Reconstituted by a Number of Pathways

Acid-induced and guanidine hydrochloride (GdnCl)-induced reversible unfolding ofEscherichia colialkaline phosphatase (AP) was characterized under equilibrium conditions. The protein was exposed to extreme conditions of pH 2.0 or 6MGdnCl and was subsequently returned to normal conditions. Associated changes in the protein structure was probed by various spectroscopic methods. The changes in the functional properties were monitored by measuring enzymatic activity, capacity to renature spontaneously upon removal of the denaturant, and renaturation in presence of various site-specific and nonspecific effector molecules, in the absence and presence of β-mercaptoethanol. Analysis of the fluorescence and CD spectra showed that the unfolding of the organized structures was much more extensive in 6MGdnCl than at pH 2.0. Intrachain S–S bonds in each unfolded state were accessible to reduction by β-mercaptoethanol. The effectors Zn2+and ATP induced renaturation of active site only under reducing conditions, whereas Triton X-100 or α-crystallin needed the presence of some organized structure. The reconstituted protein from each denatured state without or with an effector 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.