The Active Site of Pancreatic Porcine Elastase: Specificity, Size and Stereospecificity

The kinetic parameters of pancreatic‐porcine‐elastase catalyzed hydrolysis of peptides and peptide esters were determined. The most efficient and specific cleavage occurred at the Ala‐Lys bond of the S‐peptide (RNAse P_1–20), RNAse P_1–8 octapeptide and the synthetic heptapeptide Ala₅‐Lys‐Phe. The active site is extended over eight subsites (about 29 Å) and consists of five subsites from the cleaved bond toward the N‐terminal end and three subsites from the scissil bond toward the C‐terminal end of the substrate. The size of the active center was deduced by comparing the kinetic parameters of the appropriate diastereomers of peptides (of the general formula Ala₄‐Lys‐Phe and Ala_s‐Lys‐Phe), and p‐nitrobenzyl esters of tri‐ tetra‐ and penta‐alanine peptides. Specific local interactions of the various subsites, and their stereospecificity were determined by comparative study of the steady‐state kinetic parameters of the appropriate group‐pairs of substrates. Alignment of the substrate Ala₅‐Lys‐Phe‐Ala at the active site was determined by constructing the CPK (space filling model) of elastase, based on its known coordinates, determined at 2.5 Å resolution.