DENATURATION OF URIDINE PHOSPHORYLASE FROM ESCHERICHIA-COLI K-12 WITHGUANIDINE-HYDROCHLORIDE - KINETICS OF INACTIVATION, DISSOCIATION, ANDREACTIVATION OF THE ENZYME

Denaturation of uridine phosphorylase from Escherichia coli K-12 by gu anidine hydrochloride is accompanied by the displacement of the maximu m in the protein fluorescence spectrum (lambda(max)) from 331 to 348 n m. The half-maximal change in the lambda(max), position is observed at 1.18 M guanidine hydrochloride. For this concentration of denaturant, the sedimentation pattern consists of two boundaries, one of which co rresponds to the motion of the hexameric enzyme form (S-2O,S-w = 8.2 S ) and other represents a monomer (s(20,w) = 2.6 S). In the presence of 2 M guanidine hydrochloride the enzyme moves as a monomer. The kineti cs of inactivation of uridine phosphorylase by guanidine hydrochloride are complex (minima and maxima are observed on the kinetic curves). T he initial rate of the enzyme reactivation after dilution of the enzym e preincubated with guanidine hydrochloride is second order with respe ct to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of low-activity monomers into dimers followed by a rapid hexamer formation. The second-order rate constant for the reassociation of the enzyme is 3.0.10(4) M(-1).sec(-1) (50 mM berate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20 degre es C). Thiol groups become accessible to titration by 5,5'-dithiobis-( 2-nitrobenzoic acid) after treatment of uridine phosphorylase with gua nidine hydrochloride. Uridine and uracil inhibit the unfolding of the protein globule by guanidine hydrochloride.