The PA domain is crucial for determining optimum substrate length for soybean protease C1: structure and kinetics correlate with molecular function.

A subtilisin-like enzyme, soybean protease C1 (EC 3.4.21.25), initiates the degradation of the β-conglycinin storage proteins in early seedling growth. Previous kinetic studies revealed a nine-residue (P5-P4') length requirement for substrate peptides to attain optimum cleavage rates. This modeling study used the crystal structure of tomato subtilase (SBT3) as a starting model to explain the length requirement. The study also correlates structure to kinetic studies that elucidated the amino acid preferences of soybean protease C1 for P1, P1' and P4' locations of the cleavage sequence. The interactions of a number of protease C1 residues with P5, P4 and P4' residues of its substrate elucidated by this analysis can explain why the enzyme only hydrolyzes peptide bonds outside of soybean storage protein's core double β-barrel cupin domains. The findings further correlate with the literature-reported hypothesis for the subtilisin-specific protease-associated (PA) domain to play a critical role. Residues of the SBT3 PA domain also interact with the P2' residue on the substrate's carboxyl side of the scissile bond, while those on protease C1 interact with its substrate's P4' residue. This stands in contrast with the subtilisin BPN' that has no PA domain, and where the enzyme makes stronger interaction with residues on the amino side of the cleaved bond. The variable patterns of interactions between the substrate models and PA domains of tomato SBT3 and soybean protease C1 illustrate a crucial role for the PA domain in molecular recognition of their substrates.