Copyright © 2000-2011 Mark Brandt, Ph.D. 27 Enzyme Mechanisms Enzymes function by a wide variety of mechanisms. We will cover a few examples to illustrate the means that enzymes use to catalyze reactions. Serine proteases Peptide bond hydrolysis is a very common process. A wide variety of enzymes can perform proteolytic reactions. Members of one large family of protease are called “serine proteases” because of the important serine at the active site. All of the serine proteases contain three residues at their active site: a serine, a histidine, and an aspartate. These comprise the characteristic catalytic triad. In the numbering scheme for chymotrypsin (a numbering scheme which is typically used in studies of any of the mammalian serine proteases), the residues are Ser195, His57, and Asp102. Note that these residues are distributed throughout the 240 amino acid residues found in chymotrypsin, but are located in close proximity in the active three-dimensional conformation of the enzyme (see figure, below). The serine proteases are synthesized as larger, inactive, precursors. As an example: chymotrypsinogen is converted to chymotrypsin by two cleavage processes involving excision of residues 12-15 and 147-148. Interestingly the structures of chymotrypsinogen and chymotrypsin (including the catalytic residues) are almost completely superimposable; the conformational change involved in the conversion process appears to be fairly small. The implication is that relatively small structural changes can cause dramatic changes in activity. (Note: some changes in other aspects of the active site are observed. However, because the catalytic triad residues are observed in their normal conformation, the zymogen forms of serine proteases usually exhibit low levels of catalytic activity.) Asp102His57Ser195Residues147-148Residues12-15This figure shows superimposed structures of chymotrypsin (in blue, from PDB ID 1CA0) and chymotrypsinogen (in red, from PDB ID 2CGA). Note the overall similarity in the structures, especially on the left side of the figure and in the position of the catalytic triad residues. The location of the excised residues (12-15 and 147-148) removed in the conversion of chymotrypsinogen to chymotrypsin is shown in orange.Copyright © 2000-2011 Mark Brandt, Ph.D. 28 Some bacterial serine proteases (e.g., Streptomyces griseus Protease A) are structurally similar to the mammalian enzymes; the invention of the serine protease structure therefore occurred prior to the divergence of prokaryotes and eukaryotes. On the other hand, the Bacillus subtilus protease subtilisin, although it has the same catalytic residues, has a completely different structure, suggesting that serine protease mechanism has been invented more than once during evolution. The serine proteases differ in their sequence and in their substrate specificity: the bacterial protease subtilisin (one of the major enzymes that resulted in the advertising slogan “the cleaning power of enzymes!”) will cleave essentially any substrate, while one of the enzymes in the clotting cascade, Factor Xa, requires a four residue recognition sequence, Ile-Glu-Gly-Arg, in order to uniquely hydrolyze its polypeptide substrate after the arginine. Trypsin is specific for cleavage after lysine and arginine. Trypsin contains Asp189 in the substrate side-chain binding pocket, and this residue forms an electrostatic interaction with basic residues. However, the specificity is not completely dependent on a single residue; mutating Asp189 to serine (the corresponding residue in chymotrypsin) converts trypsin into a non-specific protease, rather than into an enzyme with chymotrypsin-like specificity. (Even incorporating other mutations in nearby residues is only partially effective at producing a chymotrypsin-like enzyme; proteins are complex entities!) Catalytic mechanism of serine proteases Serine proteases increase the rate of peptide bond hydrolysis by ~1010 compared to the uncatalyzed reaction. A variety of structural features are responsible for the catalytic effectiveness of these enzymes. As mentioned above, the serine proteases all have three residues that are critical for catalysis: a serine, a histidine, and an aspartic acid. These are conserved in all of the serine proteases, and are superimposable in the crystal structures of these proteins. Steps in the catalytic process: 1) Substrate binding. Note that for the substrate peptide, the side-chain of the amino acid residue immediately before the scissile peptide bond can bind to the recognition site on the enzyme. NHNHCPeptideR HNHOPeptideH ROHNNHSer195His57Asp102OOSubstrate side-chainrecognition siteCopyright © 2000-2011 Mark Brandt, Ph.D. 29 2) Nucleophilic attack. Serine195 acts as a nucleophile, facilitated by Histidine57, which abstracts a proton from Ser195. The result of the nucleophilic attack is a covalent bond between the Ser195 side-chain oxygen and the substrate. The negative charge that develops on the peptide carbonyl oxygen is stabilized by hydrogen bonds formed from two protease backbone amide protons. This region of the protein is called the “oxyanion hole”, because it stabilizes the negative charge on the oxygen; the oxyanion hole is critical for catalysis. (Note: the oxyanion hole includes the backbone amide proton from Ser195; the diagrams illustrating this discussion are thus somewhat distorted from the three-dimensional structure.) 3) Protonation. His57 donates a proton to the substrate amide nitrogen, allowing release of the C-terminal part of the substrate as a free peptide. 4) Ester hydrolysis. The final step is an attack by water on the ester bond between the peptide and the Ser195 oxygen. This forms the second product peptide with a normal carboxyl group, and regenerates the serine hydroxyl. The second peptide then dissociates from the enzyme to allow another NHNHCPeptideR HNHOPeptideH ROHNNHSer195His57Asp102OOOxyanion holeNHNHCPeptideR HNHOPeptideH RONNHSer195His57Asp102OOOxyanion holeHNHNHCPeptideR