Enzyme Mechanisms - Serine Proteases
Part V
Part V. Inhibitors (Non-Competitive Irreversible) of the Serine Proteases.
Now study the inhibitors of the enzyme. Use inhibitors to identify AAs at the active site involved in accelerating the bond rearrangements in the reaction catalyzed by the enzyme. Inhibitors which looked like substrate- but were reactive: "Active-Site Directed" inhibitors were used to identify AA side chains at the active site.
For example, Chymotrypsin is inhibited by "TPCK"
Figure 13. Structure of TPCK which is an active-site directed inhibitor of chymotrypsin since it has the required aromatic side chain of the target amino acid which directs its binding to the active site. But it does not have an amide bond to hydrolyze; instead it has a ketone in its place. In addition, on the end it has an reactive chloro-group which can be displaced by a nucleophile in the enzyme's active site attacking the carbon holding the chloro-group. This lead to the identification of His-57 as a strong nucleophilic group in the active site of chymotrypsin. Since chymotrypsin lost all enzymatic activity when reacted with TPCK, it was clear that His-57 was essential for enzyme activity. In addition, this same His-57 was found in trypsin and elastase, which suggests that these enzymes also depend on it for catalytic activity.
The TPCK reacts with a His in Chymotrypsin's active site (His-57).
Trypsin and all serine proteases are inhibited by a very reactive and toxic chemical called DFP (diisopropyl fluorophosphate), which is so toxic that one drop on your skin will kill you. It does not kill you by inhibiting serine proteases, but because it is also an inhibitor acetylcholine esterase, which is an enzyme important in nerve transmissions.
Figure 14. Structure of DFP.
DFP (Diisopropylfluorophosphate) which reacts with a serine at the active site of trypsin, chymotrypsin and elastase. This has been identified as Ser-195. Clearly, DFP has a carbonyl carbon which can be attacked by the activated serine hydroxyl of Ser-195 and since the fluorine is an excellent leaving group, the DFP derivatizes the enzyme at Ser-195. This reaction is thought to be like the one that takes place during catalysis of amide hydrolysis by serine proteases. While it is easy for the intact serine (Ser-OH) to react with DFP since it is so reactive, it was hypothesized if the same type of reaction takes place during catalysis of hydrolysis of less reactive substrates like proteins, peptides, simples amides and simple esters, then the serine must be activated.
Since it was already know that His-57 is essential for catalysis by serine proteases (see above discussion on reaction of TPCK with chymotrypsin), this led to the idea that the His-57 might be involved with activating the serine hydroxyl to make it more reactive. As you might recall from the first part of this course, the hydroxyl on serine is not ionizable since it has a pK around 16 or 18. So normally to make a hydroxyl group into its ionic form requires an redox reaction like one where sodium metal reacts with an alcohol (R-OH) to make an alkyl-oxyanion (R-O-). But in the active site of the serine protease, there is a special environment so that His-57 can act as base and pull the hydrogen off the hydroxyl group of Ser-195 as a proton which results in the conversion of Ser-OH to Ser-O-, which would be the activated form of Ser-195. Thus, this conversion of Ser-195 to a more reactive form could account for the reaction during catalysis of the less reactive substrates like amides and esters. Note in this case, the Ser-195 hydroxyl is converted to its conjugate base by ionization rather than a redox reaction.
Since it was thought by the investigators studying the serine protease catalytic mechanism that His-57 might not be strong enough by itself to pull the proton off the hydroxyl group on Ser-195, an additional group in the active site of these enzymes might act in concert with His-57 to help pull off the proton of Ser-195. Logically, an acidic group (from an Asp or Glu) could in the active site could help by taking a proton of His-57 in its charged form and this would make His-57 ready to accept the proton from Ser-195 during its activation. Unfortunately, acidic side chains like Asp and Glu have are difficult to get to react with inhibitors like were used to identify His-57 and Ser-195. The amino acid sequence comparison of the serine protease family of enzymes were helpful, but in the end the investigators had to use the 3-D models of these enzymes to come up with the best suggestion for an Asp or Glu residue in the active site most likely to fulfill this role in the mechanisms of catalysis by serine proteases. Finally, Asp-102 was identified as participating in catalysis by 'docking' model substrates to 3-D models in computers. In addition, His-57, Asp-102 and Ser-105 have all been shown to be essential amino acid side chains for catalysis by serine proteases by the newer molecular biology process of "Site-directed mutagenesis" where the key amino acids are replaced by making mutants of the enzymes and expressing them in a bacterium (see last section of this lecture for more details).
©Wilbur H. Campbell, 1995; wcampbel@mtu.edu