Denaturation of uridine phosphorylase from Escherichia coli K-12 with guanidine hydrochloride: kinetics of inactivation, dissociation, and reactivation of the enzyme.
Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride is accompanied by the displacement of the maximum in the protein fluorescence spectrum (lambda max) from 331 to 348 nm. 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 corresponds to the motion of the hexameric enzyme form (s20,w = 8.2 S) and other represents a monomer (s20,w = 2.6 S). In the presence of 2 M guanidine hydrochloride the enzyme moves as a monomer. The kinetics of inactivation of uridine phosphorylase by guanidine hydrochloride are complex (minima and maxima are observed on the kinetic curves). The initial rate of the enzyme reactivation after dilution of the enzyme preincubated with guanidine hydrochloride is second order with respect 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 borate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20 degrees C). Thiol groups become accessible to titration by 5,5'-dithiobis-(2-nitrobenzoic acid) after treatment of uridine phosphorylase with guanidine hydrochloride. Uridine and uracil inhibit the unfolding of the protein globule by guanidine hydrochloride.
Burlakova
A A
AA
State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
Kurganov
B I
BI
Chernyak VYa
Debabov
V G
VG
D004793:Enzyme Reactivators / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D004793:Enzyme Reactivators; D006146:Guanidines; D014543:Uridine Phosphorylase; D019791:Guanidine