Enzyme Mechanisms - Serine Proteases
Part IV
Part IV. Artificial Substrates for Trypsin and Chymotrypsin.
In addition to catalyzing hydrolysis of amides in proteins and peptides, these enzymes can also hydrolyze artificial substrates, such as simple amides and esters (see Fig. 10), which were very helpful in working out the mechanism of catalysis since the simple substrates are easy to analyze as compared to complex substrates like proteins and peptides. The key thing about the simple substrates is that they have the same or similar side chains as are found in the standard peptide/protein substrates of trypsin and/or chymotrypsin so that they bind to the active site. The fact that these simple amide and ester substrates are hydrolyzed in the reactions catalyzed by trypsin and chymotrypsin is in itself informative because it shows that catalysis by these enzymes is a general process where the key fact is that substrate must have a carbonyl group susceptible to attack by the reactive parts of the enzyme. The simple substrates not only are more easily studied than protein or peptide substrates, but also react more rapidly. This attracted more 'chemical' type of scientist to the study of these enzymes and the mechanism of catalysis was then studied by conventional chemical methods rather just biochemical methods. An example of this approach is where the investigators went beyond standard 'steady-state' enzyme kinetics studies and studied the events which happen before the 'steady-state' is established, which is logically called 'pre-steady-state' kinetic analysis (see Fig. 11).
Figure 10. Simple amides and esters are artificial substrates for trypsin and chymotrypsin.
The steady state kinetic analysis for Km and Vmax gave different results for amides than esters. So pre-steady state (or fast reaction) kinetics were used to analyze these substrate kinetic patterns.
Figure 11. Apparatus for doing fast-reaction or pre-steady state kinetic analysis. The substrate and enzyme are pushed together into the reaction chamber where observations of the initial events in catalysis are watched using a spectrophotometer and recorded. The reaction can also be stopped automatically by triggering the stop syringe into action.
The simple amide and ester substrates are very useful for kinetic analysis. From the studies of the 'pre-steady-state' kinetics it was learned that the reaction takes place in 2 steps:
1. Enzyme attacks substrate forming an Enz-Acyl intermediate
2. Enz-Acyl intermediate is hydrolyzed with H2O.
This helped to show that the enzyme formed a covalent bond with substrate
1. For amides, binding of substrate and formation of the Enz-Acyl bond is rate limiting
2. For esters, binding and formation of Enz-Acyl bond is rapid, while hydrolysis of the Enz-Acyl intermediate is slowest.
Figure 12. Mechanism for chymotrypsin catalyzed hydrolysis of a simple amide.
©Wilbur H. Campbell, 1995; wcampbel@mtu.edu