Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin
Authors: FIEDLER Franz 
Published: March 2005

Kinetic constants for the hydrolysis by porcine tissue β‐kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions.

k
cat and kcat/Km values for the hydrolysis of the Arg‐Ser bonds of the peptides by trypsin are conspicuously high. kcat for the best of the peptide substrates, Ac‐Phe‐Arg‐Ser‐Val‐NH2, even reaches kcat for the corresponding methyl ester, indicating rate‐limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. kcat/Km for the hydrolysis of the peptide esters with different nonpolar L‐amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. kcat for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue.

In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate‐limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least S′3 of tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly‐216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well.

In sharp contrast to the behaviour of trypsin is the very strong influence of the nature of the P2 residue in tissue‐kallikrein‐catalyzed reactions. kcat/Km varies 75‐fold in the series of the dipeptide esters with nonpolar L‐amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes kcat/Km as much as 730‐fold. This behaviour, together with the high value of kcat/Km for Ac‐Phe‐Arg‐OMe of 3.75 × 107 M−1 s−1, suggests rate‐limiting binding (k1) in the hydrolysis of the best ester substrates. The P2 residue mainly affects Km. The effects of the nature of the P2 residue on the hydrolysis of the dipeptide esters are in accordance with the proposed sandwiching of P2 residues between Tyr‐99 and Trp‐215 of tissue kallikrein. The pronounced secondary specificity of subsite S2 for bulky, hydrophobic amino acids appears to be a general feature of tissue kallikreins. Remarkably, the two most favourable P2 residues, phenylalanine and leucine, also occur in this position at the two tissue kallikrein cleavage sites of the natural substrate, kininogen. A comparison of the kinetic constants of the kininogen peptides with those for kallidin release from kininogen indicates that still further interactions with other parts of the kininogen molecule play a role in the cleavage at the kininogen Arg site by tissue kallikrein.