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<StructureSection load='' size='350' side='right' scene='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/2' caption='α-chymoptrypsin (PDB code [[2cha]])'> == '''Introduction to Serine Proteases''' == '''Serine proteases''' account for over one-third of all known proteolytic enzymes <ref>PMID:17991683</ref>,<ref name="DiCera">PMID:19180666</ref>. 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) <ref name ="DiCera"/>, 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 [http://www.expasy.org/ ExPASy Proteomics Server] under the [http://www.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html 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 <ref>PMID: 7795518</ref>. 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'''''! *<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/2'>Chymotrypsin</scene> *<scene name='User:Amy_Kerzmann/Sandbox_5/New_trypsin-wt-triad/4'>Trypsin</scene> *<scene name='User:Amy_Kerzmann/Sandbox_5/New_elastase-triad/3'>Elastase</scene> For a side-by-side comparision of these three structures, see an [[User:Amy_Kerzmann/Sandbox_5|alternate version of this article]]. == '''Active Sites''' == Serine proteases perform their catalytic roles using three key residues, which are commonly referred to as the '''catalytic triad''': Ser, His, Asp. Highlight the <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/7'>chymotrypsin catalytic triad</scene>. The elements are color coded as follows: {{Template:ColorKey_Element_C}}, {{Template:ColorKey_Element_O}}, {{Template:ColorKey_Element_N}}. * Mouse over or click on the structure to determine the residue numbers for Ser, Asp and His. (The residue code will appear near the mouse pointer or in the lower left-hand corner of the browser window.) * You can adjust the zoom in each image by holding down the shift key while you click and drag on the structure. Alternatively, you can click on the Jmol symbol in the lower right-hand corner of each image and select a different zoom percentage from the main menu. This arrangement of amino acids is also called a '''charge relay system''' <ref>PMID: 7016210</ref>. 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? ([http://prowl.rockefeller.edu/aainfo/volume.htm pKa table]) Now compare the active site residues of chymotrypsin to the <scene name='User:Amy_Kerzmann/Sandbox_5/New_trypsin-wt-triad/10'>trypsin catalytic triad</scene> and the <scene name='User:Amy_Kerzmann/Sandbox_5/New_elastase-triad/8'>elastase catalytic triad</scene>. == '''Substrate Binding Pockets''' == As 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: {{Template:ColorKey_Hydrophobic}} or {{Template:ColorKey_Polar}}, with <font color="FF0000">'''Aspartate'''</font> highlighted further ). *The <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/4'>chymotrypsin binding pocket</scene> is large, deep and relatively hydrophobic. This structure accommodates bulky aromatic and aliphatic sidechains, as indicated by the position of a <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/6'>p-sulfinotoluene</scene>, a bound inhibitor. *The <scene name='User:Amy_Kerzmann/Sandbox_5/New_trypsin-wt-triad/6'>trypsin binding pocket</scene> contains <font color="FF0000">Asp189</font> to select for positively charged sidechains, such as <scene name='User:Amy_Kerzmann/Sandbox_5/New_trypsin-wt-triad/8'>arginine</scene>. The arginine sidechain is part of a larger peptide-based inhibitor called <scene name='User:Amy_Kerzmann/Sandbox_5/New_trypsin-wt-triad/9'>aeruginosin 98-B</scene>, which is now shown in balls and sticks. *The <scene name='User:Amy_Kerzmann/Sandbox_5/New_elastase-triad/6'>elastase binding pocket</scene> is more constrained, explaining the preference for smaller residues. Which residue provides the key steric hinderance to prevent larger sidechains from entering the binding pocket? == '''Disulfide Bonds''' == These 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 <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/9'>chymotrypsin</scene>, <scene name='User:Amy_Kerzmann/Sandbox_5/New_trypsin-wt-triad/11'>trypsin</scene> and <scene name='User:Amy_Kerzmann/Sandbox_5/New_elastase-triad/9'>elastase</scene>. 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 Structures''' == In 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 [http://www.pdb.org/pdb/explore/explore.do?structureId=1GZX 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 [http://www.pdb.org/pdb/home/home.do RSCB Protein Data Bank], here are links to each of the crystal structures shown above: [http://www.pdb.org/pdb/explore/explore.do?structureId=2cha chymotrypsin (2cha)], [http://www.pdb.org/pdb/explore/explore.do?structureId=1aq7 trypsin (1aq7)] and [http://www.pdb.org/pdb/explore/explore.do?structureId=4est elastase (4est)]. Keep in mind that these are only representative structures of each serine protease. Other structures can be found at the following links: * [http://www.pdb.org/pdb/explore/explore.do?structureId=7GCH chymotrypsin (7gch)] This structure is shown in Figure 6-18 (page 206) of [http://www.whfreeman.com/newcatalog.aspx?search=Lehninger&isbn=071677108X Lehninger's Principles of Biochemistry (5th edition)]. * [http://www.pdb.org/pdb/explore/explore.do?structureId=1acb chymotrypsin (1acb)] This structure includes a bound protein, showing how the peptide fits into the active site of the enzyme. * [http://www.pdb.org/pdb/explore/explore.do?structureId=2cmy trypsin (2cmy)] * [http://www.pdb.org/pdb/explore/explore.do?structureId=3est elastase (3est)] ==3D structures of chymotrypsin== [[Chymotrypsin]] ==3D structures of trypsin== [[Trypsin]] ==3D structures of elastase== [[Elastase]] ---- == '''Understanding the Mechanism''' == ==== '''Catalytic Mechanism''' ==== [http://www.whfreeman.com/newcatalog.aspx?search=Lehninger&isbn=071677108X Lehninger's Principles of Biochemistry (5th edition)] describes the catalytic mechanism of chymotrypsin on pages 208-209. An [http://bcs.whfreeman.com/lehninger5e/pages/bcs-main.asp?v=&s=06000&n=00010&i=06010.01&o=|00610|00580|00590|00510|00540|00600|00550|00570|00630|00010|00020|00030|00040|00070|00080|00090|00100|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|24000|25000|26000|27000|28000|99000| animated version] of the enzyme-catalyzed hydrolysis reaction is also available on the textbook's website. <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/10'> This representation</scene> was designed to match the perspective given by those resources. To provide better orientation after this rotation, here are the <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/11'>binding pocket residues</scene> that were highlighted above. (Or you can <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/16'>label the catalytic triad and Gly193</scene>.) *<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/12'>show p-sulfinotoluene binding</scene> *<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/13'>show just p-sulfinotoluene</scene> (<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/14'>as sticks</scene>) Note that the <scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/15'>sulfino group</scene> would be in approximately the same location as the carbonyl group of the substrate peptide. *<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/10'>hide binding pocket</scene> ==== '''pH Dependence''' ==== The 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: *<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/23'>show binding pocket residues with Ile16 (spacefill, atomic coloring)</scene> *<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/22'>show binding pocket with p-sulfinotoluene and Ile16 (spacefill, atomic coloring)</scene> *<scene name='User:Amy_Kerzmann/Sandbox_5/New_chymotrypsin-triad/19'>show p-sulfinotoluene with Ile16 (sticks)</scene> 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 <ref>Bender ML, Kézdy FJ. The Current Status of the α-Chymotrypsin Mechanism. J. Amer. Chem. Soc. 1964; 86(18):3704–3714. [http://pubs.acs.org/doi/abs/10.1021/ja01072a020 Bender & Kézdy]</ref>. 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== <references />

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