Binding of Nonphysiological Protein and Peptide Substrates to Proteases:  Differences between Urokinase-Type Plasminogen Activator and Trypsin and Contributions to the Evolution of Regulated Proteolysis

Abstract

Understanding the regulation of physiological processes requires detailed knowledge of the recognition of substrates by enzymes. One of the most productive model systems for the study of enzyme-substrate interactions is the serine protease family; however, most studies of protease action have used small substrates that contain an activated, non-natural scissile bond. Because few kinetic or structural studies have used protein substrates, the physiologically relevant target of most proteases, it seems likely that important mechanisms of substrate recognition and processing by proteases have not yet been fully elucidated. Consistent with this hypothesis, we have observed that K(m) values for protein substrates are reduced as much as 200-15000-fold relative to those of analogous peptide substrates. Here we examine the thermodynamic consequences of interactions between proteases and their substrates using staphylococcal nuclease (SNase) and SNase variants as model protein substrates. We have obtained values for enthalpy, entropy, and K(d) for binding of proteins and peptides by the nonspecific protease trypsin and the highly specific protease urokinase-type plasminogen activator (u-PA). To avoid cleavage of substrates during these measurements, we used inactive variants of trypsin and u-PA whose catalytic serine S195 had been replaced by alanine. Differences in the K(d) values for binding of protein and peptide substrates closely approximate the large differences observed in the corresponding K(m) values. Improved binding of protein substrates is due to decreased enthalpy, and this effect is pronounced for the selective protease u-PA. Fundamental differences in recognition of analogous protein and peptide substrates may have influenced the evolution of protease specificity.

Identifi cation of protease exosite-interacting peptides that enhance substrate cleavage kinetics

“…There are now multiple examples of proteases that cleave physiological protein substrates orders of magnitude faster than the best known active site interacting peptide substrate. Urokinase-type plasminogen activator, for example, can cleave the physiological substrate plasminogen more than 100-fold faster than the best known peptide substrates selected using substrate phage display (e.g., PFGR↑SA) (Bergstrom et al, 2003). Although the precise mechanisms used by proteases to cleave physiological substrates so efficiently remain unclear, a subset of proteases can recognize an extended linear peptide substrate using multiple interacting exosites in a manner similar to thrombin.…”

Kinetic mechanism for p38 MAP kinase α

“…There is currently a lot of interest in understanding the molecular recognition events associated with MAPKs, because docking domains are thought to play a major role in determining the specificity of substrate-ligand and protein-ligand interactions [13][14][15]. A growing number of enzymes are thought to utilize docking domains, which are substrate recognition elements lying outside the active site of the enzyme and which govern the formation of an enzyme-substrate complex [16][17][18][19][20][21][22][23]. Several years ago, we showed that despite the presence of docking domains on p38 MAPKa, which could tether a protein substrate and facilitate multiple phosphorylations in one collision, p38 MAPKa phosphorylates ATF2D115 on Thr69 and Thr71 in a nonprocessive manner [24].…”