Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin: Sites of cleavage and activity of the modified enzyme

Abstract

Subtilisin catalyzes a limited modification of rabbit liver fructose 1,6-bisphosphatase (Fru-P 2ase, 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 NH 2-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 NH 2-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.