Fructose 1,6-bisphosphatase (Fru-P2ase,EC 3.1.3.11) purified from livers of fed rabbits has been reported to contain tryptophan, which is not present in the enzyme purified from livers of fasted animals. We now show that the tryptophan arises from small amounts of active or inactive rabbit liver aldolase in the Fru-P2ase preparations. Fru-P2ase free of tryptophan may conveniently be prepared by raising the temperature of the heat step in the purification procedure to 67 °C.

A cyanogen bromide peptide, isolated from the NH2-terminus of rabbit liver FruP2ase, has been shown to have the following structure: Acetyl-Ala-Asp-Lys-Ala-Pro-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met. Previous evidence that the enzyme contains tryptophan located near the NH2-terminus and also that this tryptophan is lost on exposure of the enzyme to lysosomal fractions must be reevaluated. It is unlikely that the NH2-terminus would be reacetylated following proteolytic modification of this portion of the molecule.

Subtilisin catalyzes a limited modification of rabbit liver fructose 1,6-bisphosphatase (Fru-P2ase, EC 3.1.3.11), resulting in enhanced catalytic activity measured at pH 9.2. Sequence analysis of a cyanogen bromide peptide containing the subtilisin cleavage sites and of the NH2-terminal portion of the residual subunits shows that four peptide bonds in a limited region of the native molecule are susceptible to subtilisin. The cleavage sites are located at peptide bonds 57–58 (tyrosine-glycine), 60–61 (alanineglycine), 63–64 (threonine-asparagine), and 66–67 (threonine-glycine). Cleavage of the threonine-glycine bond appears to occur in about 60% of the peptide chains, and cleavage of the tyrosine-glycine bond occurs to a limited extent only at higher ratios of subtilisin to fructose bisphosphatase. The results suggest that the region of the molecule including residues 57–67 may exist as an exposed peptide susceptible to proteolytic attack. The residual subunits have been separated from the S-peptide (residues 1–60) and renatured in the presence of dithiothreitol. The reconstituted tetrameric enzyme lacking residues 1–60 is catalytically active. Thus, the NH2-terminal portion of the molecule is not required for this activity. The sequence of residues 1–78 of rabbit liver fructose 1,6-bisphosphatase has now been determined.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin followed by denaturation of the protein yields a peptide containing 60 amino acid residues, including the blocked NH2-terminus. This peptide has the following sequence: Ac-Ala-Asp-Lys-Ala-Pr o-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met-Glu-Glu-Gly-Arg-Ly s-Ala-Gly-Gly-Thr-Gly-Glu-Met-Thr-Gln-Leu-Leu-Asn-Ser-Leu-Cys-Thr-Ala-Va l-Lys-Ala-Ile-Ser-Thr-Ala-Val-Arg-Lys-Ala-Gly-Ile-Ala-His-Leu-Tyr-Gly-Ile-Ala.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin results in a several-fold increase in catalytic activity measured at pH 9.2. This change is due to cleavage of a peptide bond located 60 amino acid residues from the NH2-terminus. The S-peptide and the residual subunit appear as separate peptides in sodium dodecyl sulfate polyacrylamide gel electrophoresis and the S-peptide can be isolated by gel filtration in 9% HCOOH. Under nondissociating conditions, however, the S-peptide remains associated with the protein, and the tetrameric structure and original molecular weight are preserved. Thus the nicking of the peptide chain by subtilisin causes a conformation change that alters the catalytic properties of the enzyme.