Serine Proteases
From Proteopedia
Introduction to Serine ProteasesSerine proteases account for over one-third of all known proteolytic enzymes [1],[2]. Within the diverse collection of serine proteases, the most famous members are trypsin, chymotrypsin and elastase. Aside from their key roles in digestion (and other physiological processes) [2], the unique specificities of these enzymes make them useful tools in biochemistry and molecular biology to ascertain protein sequences. More information about utilizing proteases for research can be found on the ExPASy Proteomics Server under the PeptideCutter Tool.
Looking at the structures below, it is apparent that these three enzymes have similar folds. This conservation of tertiary structure is due to extensive similarities at the level of primary amino acid sequence. However, there are small differences in amino acid sequence among the proteins, which are reflected in their different specificities. Each protein cleaves the peptide backbone after (or on the carbonyl side) of a specific type of sidechain; chymotrypsin prefers to cut after aromatic residues, trypsin after basic residues and elastase after smaller neutral residues [3]. After examining the molecular basis for these functional similarities and differences, you will hopefully see why serine proteases are a classic example of how structure dictates function! For a side-by-side comparision of these three structures, see an alternate version of this article. Active SitesSerine proteases perform their catalytic roles using three key residues, which are commonly referred to as the catalytic triad: Ser, His, Asp. Highlight the . The elements are color coded as follows: C, O, N.
This arrangement of amino acids is also called a charge relay system [4]. Considering that the serine sidechain becomes activated for catalysis when it is negatively charged, how would the protons move among the highlighted Ser, Asp and His residues? Are these proton exchanges what you would expect from your knowledge of the pKa values for these amino acid sidechains? (pKa table) Now compare the active site residues of chymotrypsin to the and the . Substrate Binding PocketsAs we have just seen, these three serine proteases have relatively similar active sites. What then accounts for their varying specificities? To answer this question, the next links examine the binding pockets of each protein. The spacefilled residues have been color coded according to hydrophobicity (residues are indicated as: Hydrophobic or Polar, with Aspartate highlighted further ).
Disulfide BondsThese proteases each have four to six disulfide bonds. One cystine linkage that is conserved among all the structures is between Cys191 and Cys220. These residues were shown in spacefill representation under the "Substrate Binding Pockets" heading, but are more easily viewed in sticks format as can be seen here for , and . What role might this covalent bond have in the protein's function? Does its conservation among several proteins with similar function provide any suggestion to its importance? Additional PDB StructuresIn order to easily compare the proteins shown on this page, some portions of the crystal structures have been masked. Although each of these serine proteases functions as a monomer, they are often observed as dimers or even tetramers in crystal structures. These higher-order multimers are not the physiological state of the serine protease, but rather a consequence of the experimental method, which requires high protein concentrations. However, some proteins are only functional in the tetrameric state, such as hemoglobin. Therefore, it is important to recognize that one cannot necessarily determine the physiological state from a crystal structure alone. To view the full, unmodified structures in the RSCB Protein Data Bank, here are links to each of the crystal structures shown above: chymotrypsin (2cha), trypsin (1aq7) and elastase (4est). Keep in mind that these are only representative structures of each serine protease. Other structures can be found at the following links:
3D structures of chymotrypsin3D structures of trypsin3D structures of elastaseUnderstanding the MechanismCatalytic MechanismLehninger's Principles of Biochemistry (5th edition) describes the catalytic mechanism of chymotrypsin on pages 208-209. An animated version of the enzyme-catalyzed hydrolysis reaction is also available on the textbook's website. was designed to match the perspective given by those resources. To provide better orientation after this rotation, here are the that were highlighted above. (Or you can .)
pH DependenceThe pH dependence of chymotrypsin activity is a well-studied phenomenon. Enzymatic activity is greatest when the solution pH is between 7 and 8.5 due to the ionization states of two key residues: His57 and Ile16. If you are wondering how the isoleucine sidechain can be titrated, good question! The sidechain is not being ionized, but rather the backbone; Ile16 falls at the N-terminus of one polypeptide chain (see Lehninger, page 206). Although this representation of chymotrypsin has been uniformly colored green, the protein is actually comprised of three polypeptide chains. Now closely examine the distance between the substrate and the N-terminal nitrogen of Ile16: Ile16 appears to be too far from the active site to influence substrate binding directly, doesn't it? However, the protonated form of Ile16 is capable of forming a salt-bridge with Asp194, which opens the substrate-binding pocket [5]. Conversely, when Ile16 is deprotonated at high pH, it can no longer interact with Asp194, thereby allowing Asp194 to shift position and obstruct the substrate from entering the active site. The 'closed' conformation is further stabilized by a hydrogen bond between Asp194 and His40. In this manner, Asp194 acts as a gate for the substrate-binding pocket, with the ionization state of Ile16 acting as the latch. References
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