Fructose 1,6‐bisphosphate aldolase from liver: The absolute configuration of the intermediate carbinolamine

Introduction The classical studies of Rose et al. [ 1,2] on the stereochemistry of the aldolase reaction have shown that the enzyme controls the orientation of the alde- hyde approaching the active site as well as which face (face si) of the enolate ion is attacked. We complete here the description of the configura- tion of the enolate ion by proposing that the lysyl amino group of the active site [3 J attacks the ‘face si’ of the C-2 group of fructose bisphosphate with the intermediate formation of the (2R)-carbinolamine. 2. Materials and methods Fructose bisphosphate aldolase (D-fructose 1,6- bisphosphate D-glyceraldehyde 3-phosphate lyase, EC 4.1.2.13) from beef liver was prepared according to the procedure of Chappel et al. [4]. Only form II (spec. act. 0.8 I U/mg protein) was used in these experiments. Aldolase activity was measured spectro- photometrically [S] . Protein was measured assuming an absorbance of 0.89 at 280 nm for a 1 mg/ml solu- tion of liver aldolase [6]. [U-“C] Fructose bisphos- phate was purchased from the Radiochemical Centre, Amersham, England. N6-&Glycerollysine was prepared according to the procedure of Speck et al. [7]. N6 (2-Deoxy-2-glucitol) L-lysine (glucitollysine) and N6 (2-deoxy-2-mannitol) Llysine (mannitollysine) were prepared by condensa- tion of bromobutylhydantoin with either mannitol- amine or glucitolamine. The procedure was the same as for glycerollysine except that condensation was performed in methanol. 244 Mannitollysine, glucitollysine and glycerollysine were separated by chromatography on a Dowex 50-X8 (Na’-form) 200-400 mesh 1.1 X 80 cm column follow- ing the procedure of Moore and Stein [8]. The radioactive protein derivative was precipitated by the addition of 436 mg solid ammonium sulfate/ ml of solution. The precipitate was dissolved in 1 of M NaCI, dialysed against 0.15 M NaCl and against water. Radioactive determinations were made in a Packard Tri-Carl scintillation counter in 10 ml of Bray solution [9]. Protein hydrolysis was performed at 110°C for 48 h in evacuated sealed tubes. The hydrolysate was dried under reduced pressure in a rotary evaporator, dissolved and dried three times from water and finally dissolved in 2 ml of 0.1 M sodium citrate buffer, pH 3.42. 3. Results and discussion Liver aldolase (9 mg) was treated for 7 min at 2°C with 0.64 mM [U-14C]fructose bisphosphate (specific radioactivity 3 100 cpm/nmol) and 17 mM NaBH4 in the presence of 0.1 M sodium acetate buffer, pH 5.3. After treatment, catalytic activity was reduced to 30% and the radioactive substrate (192 000 cpm) was irreversibly bound to the enzyme. The protein was hydrolysed in 6 N HCl and the hydrolysate was submitted to chromatography on Dowex 50-X8 (Na’-form). Two radioactive peaks (fig.1) were eluted accounting for 12% and 68% respectively of the radioactivity (192 000 cpm) placed on the column. The first peak (23 000 cpm) was coincident with the authentic standard of glucitol- lysine and the second peak (130 000 cpm) was