Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase: IV. Structural features revealed by treatment with urea and Ellman's reagent

Abstract

Native 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida and three other forms generated by experimental manipulation were investigated with respect to catalytic activity, reactivity with bisdithiodinitrobenzoate (Ellman's reagent), and, in some instances, were also partially characterized by optical rotatory dispersion measurements. These include: (a) the form present in 8 m urea; (b) the aldolase reconstituted after urea treatment, and (c) the ϵ- N-(1-carboxyethyl)-substituted aldolase generated by treatment with pyruvate and NaBH 4. The native form had an apparent α-helix content of 31–33%. It reacted with 5.2 moles of Ellman's reagent per mole of aldolase without loss in activity. In the presence of 8 m urea, the α-helix content was zero and 12.1 moles of Ellman's reagent reacted per mole of aldolase. Thus, the four cysteinyl groups known to be present per subunit of the trimeric structure are distributed such that two are readily accessible and two are buried. The rate of reaction with Ellman's reagent depended upon the buffer present, as follows: HCO 3 > Tris = imidazole > arsenate ≧ phosphate > sulfate. Further, incomplete titration was observed in urea-phosphate as compared with urea-bicarbonate. After inactivation in 8 m urea-phosphate, as much as 90% of the original activity could be recovered. However, reactivation could not be achieved after treatment with urea-bicarbonate. The reconstituted aldolase behaved identically to the native aldolase by the criteria of titration with Ellman's reagent, density-gradient centrifugation, disc-gel electrophoresis, fractionation, crystal structure, α-helix content, and specific activity. Titration of ϵ- N-(1-carboxyethyl)lysyl 2-keto-3-deoxy-6-phosphogluconate aldolase with Ellman's reagent resulted in much lower reactivity of both free and buried SH groups. Analysis in urea density gradients revealed that dissociation in 8 m urea was very incomplete, thereby explaining the greater inaccessibility of thiol groups.