Isolation and properties of 6-phosphogluconate dehydrogenase from Escherichia coli. Some comparisons with the thermophilic enzyme from Bacillus stearothermophilus

Abstract

6-Phosphogluconate dehydrogenase [6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating), EC 1.1.1.44] of E. coli MRE 600 was isolated and compared with the thermostable enzyme previously isolated from B. stearothermophilus. The purified enzyme appeared homogeneous by the criteria of disc gel electrophoresis with and without sodium dodecyl sulfate, ultracentrifugation and gel filtration. The enzyme has enzymological and physicochemical properties similar to the enzyme isolated from other sources, including B. stearothermophilus. The E. coli enzyme has a MW of 100,000 .+-. 3000 and is composed of 2 apparently identical subunits. The amino acid composition of the mesophilic and thermophilic enzyme was determined and found to present large similarities. The E. coli enzyme shows a high degree of specificity for NADP, and it is inhibited by NADPH. Cysteine residues are involved in the catalytic activity, since on incubation of the enzyme with p-chloromercuribenzoate or 5,5''-dithiobis(2-nitrobenzoic acid), strong inhibition occurs, activity being restored by treatment with excess of .beta.-mercaptoethanol. The substrate 6-phosphogluconate partially protects the enzyme from inactivation. The mesophilic and thermophilic 6-phosphogluconate dehydrogenases are inactivated by Rose Bengal in the presence of light by similar kinetics and protected against photoinactivation by the enzyme substrate. The E. coli enzyme showed distinct differences in stability against heat and unfolding agents in respect to the B. stearothermophilus enzyme. Heating at 50.degree. C or incubation in 8 M urea results in rapid inactivation. The gross structure of the mesophilic and thermophilic enzyme was very similar as judged by circular dichroic [CD] measurements. The far-UV CD spectrum had a negative band centered at .apprx. 220 nm. In both cases, the fluorescence emission spectrum indicates that the environment of the tryptophan residues is similar, since both enzymes show an emission maximum at 334 nm upon excitation at 295 nm. CD measured at various temperatures between 25-80.degree. C showed the mesophilic enzyme to be conformationally stable below .apprx. 45.degree. C and the thermophilic enzyme below 60.degree. C. The secondary structure of the E. coli enzyme was very sensitive to the denaturing action of urea, since in 8 M urea it rapidly unfolded. Partial renaturation after urea treatment occurred on dilution with buffer or dialysis, as evidenced by spectral properties of the renatured enzyme. The mesophilic and thermophilic enzymes are very similar and differences in thermal stability depend on subtle differences in the architectures of the proteins.