Denaturation of Uridine Phosphorylase from Escherichia coli K-12 with Guanidine Hydrochloride: Kinetics of Enzyme Inactivation, Dissociation, and Reactivation of the Enzyme

A. A. Burlakova,^1,2 B. I. Kurganov,³ V. Ya. Chernyak,⁴ and V. G. Debabov¹

¹State Research Institute of Genetics and Selection of Industrial Microorganisms, 1-i Dorozhnyi Proezd 1, Moscow, 113545 Russia; fax: (7-095) 315-05-01.

²To whom correspondence should be addressed.

³Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia; fax: (7-095) 954-27-32; E-mail: inbio@glas.apc.org

⁴Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-03-38 ; E-mail: libro@genebee.msu.su

Submitted September 26, 1996; revision submitted October 11, 1996.

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 (s_20,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 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 3.0·10⁴ M^-1·sec^-1 (50 mM borate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20°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.

KEY WORDS: uridine phosphorylase of Escherichia coli, kinetics of inactivation with guanidine hydrochloride, dissociation--reassociation, sedimentation, fluorescence.

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