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Organic Chemistry Option II: Chemical Biology

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Organic Chemistry Option II: Chemical Biology Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Organic Chemistry Option II: Chemical Biology Dr Stuart Conway Department of Chemistry, Chemistry Research Laboratory, University of Oxford email: [email protected] Teaching webpage (to download hand-outs): http://conway.chem.ox.ac.uk/Teaching.html Recommended books: Biochemistry 4th Edition by Voet and Voet, published by Wiley, ISBN: 978-0-470-57095-1. Foundations of Chemical Biology by Dobson, Gerrard and Pratt, published by OUP (primer) ISBN: 0-19-924899-0 1 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Naturally occurring amino acids slide 66 Protein structure –α-helices slide 67 • Only one helical polypeptide conformation has simultaneously allowed conformational angles and a favourable hydrogen-binding pattern. • This striking element of secondary structure is known as the α-helix. 2 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Protein structure – anti-parallel β-sheets slide 68 O H Phe O H Phe O H N N N N N Lys O H Val O H Gln Trp H O Thr H O Gln N N N N N H O Ala H O Ile H • Anti-parallel β-sheets are an important type of protein secondary structure. • This arrangement results in a strong hydrogen bond with a near optimal N-O distance. Protein structure – parallel β-sheets slide 69 O H Phe O H Asp N N N N N H Leu O H Ile O H O H Asp O H Trp O H Ala N N N N N N Leu O H Ile O H Ile O H • β-sheets can also have a parallel arrangement. • This results in a staggered pattern of hydrogen-bonding. Protein structure – β-turns slide 70 • There are two types of β-turns, Type I and Type II. • Each comprises four key amino acids. 3 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Enzymes slide 71 • • The rates of enzymatically catalysed reactions are typically 106 to 1012 greater than the corresponding uncatalysed reactions. • • • As enzymes are chiral the active site is “chiral space” allowing differentiation between pro- chiral groups. Enzymes slide 72 • Types of enzyme catalysis: 1. 2. 3. Metal ion catalysis. 4. Electrostatic catalysis. 5. 6. Preferential binding (stabilisation) of the transition state complex. 4 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Egg white lysozyme (retaining glycosidase) slide 74 • Lysozyme enzymes are glycoside hydrolase or glycosidase enzymes. • These enzymes catalyse hydrolysis or transacetylation of glycosidic linkage in sugars. • Egg white lysozyme is a retaining glycosidase, meaning that the configuration of the anomeric centre is the same in substrate and product. • The retaining glycosidases employ both acid-base catalysis and covalent catalysis in their mechanism. • Chemical tools and crystallography have helped to determine the proposed mechanism of this enzyme. Egg white lysozyme (retaining glycosidase) slide 75 • • These enzymes work by hydrolysing β (1→4) glycosidic linkages from N-acetylmuramic acid to N-acetylglucosamine. • Lysozyme occurs mainly in the cells and secretions of vertebrate, where it may function as an antibacterial agent. • • Hen egg white lysozyme is the most widely studied species of lysosyme, mainly as it is readily available - one egg contains about 5 g. Egg white lysozyme (retaining glycosidase) slide 76 5 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Egg white lysozyme (retaining glycosidase) slide 77 Egg white lysozyme (retaining glycosidase) slide 78 retaining glycosidase retaining glycosidase retaining glycosidase E35 E35 E35 O O O O O O H H OH HO H O O R' HO HO O O O O O O D52 D52 D52 retaining glycosidase retaining glycosidase retaining glycosidase 6 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Egg white lysozyme (retaining glycosidase) slide 79 Egg white lysozyme (retaining glycosidase) slide 80 • The proposed covalent intermediate in the mechanism of lysozyme had never been observed, as the breakdown of this intermediate must be much faster than the rate of formation, in order for the enzyme to function efficiently. • A sugar containing a fluoride at the anomeric position should react rapidly with the enzyme to form a covalent intermediate. • The additional fluorine at the C2 position of the ring will reduce the rate of the covalent intermediate breaking down. • Mutation of glutamate 35 to glutamine (E35Q) slows the rate of the reaction further, meaning that the covalent intermediate with the fluorosugar accumulates and can be observed by X-ray crystallography. 7 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Inverting glycosidase slide 82 • Inverting glycosidases catalyse the same overall transformation as retaining glycosidases, but the resulting effect on the stereochemistry of the anomeric centre is different. • The mechanism of inverting glycosidases means that the configuration of the anomeric centre is inverted during the reaction. • Inverting glycosidases employ acid-base catalysis in their mechanism. • Glycosidases are important in all forms of life, mainly in the metabolism of complex carbohydrates. Inverting glycosidase slide 83 Inverting glycosidase slide 84 8 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Inverting glycosidase slide 85 inverting glycosidase inverting glycosidase inverting glycosidase E95 E95 E95 O O O O O O H OH O H O H H O O O O O O D278 D278 D278 inverting glycosidase inverting glycosidase inverting glycosidase Inverting glycosidase slide 86 9 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Serine protease slide 88 • Serine proteases (e.g. trypsin, chymotrypsin, elastase) are digestive enzymes that are synthesised in pancreatic acinar cells and secreted into the small intestine. • These enzymes all catalyse the hydrolysis of amide bonds. • Different enzymes have different selectivities for the amino acid side chains that flank the amide bond to be cleaved. • Serine proteases employ both acid-base catalysis and covalent catalysis in their mechanism. • Additionally, the intermediate oxyanion is stabilised in the “oxyanion hole”. Serine protease slide 89 10 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Serine protease slide 90 Serine protease slide 91 serine protease H57 serine protease H57 S195 S195 O N N O N N H H H 102D O 102D O O H O H N R R' O serine protease H57 serine protease H57 S195 S195 O N N O N N H H H 102D O O 102D O O O O O R R H 11 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Serine protease slide 92 Serine protease slide 93 • • The negatively charged oxygen forms hydrogen bonds with the backbone NH groups of Gly193 and Ser195. Serine protease slide 94 12 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Sulfatase slide 96 • Sulfatase enzymes cleave sulfate esters in biological systems. • These enzymes are involved in regulating the sulfation states that determine the function of may physiologically important molecules. • The mechanism of sulfatase enzymes involves covalent catalysis, metal (calcium) ion catalysis, acid-base catalysis. • In addition, one of the catalytic residues is post-translationally modified to aid the catalytic function of these enzymes. Sulfatase slide 97 Sulfatase slide 98 13 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Sulfatase slide 99 sulfatase sulfatase H H N H211 N H211 N N H H R R O O S S O O O O O O H Ca2+ H Ca2+ N N N N H O H O O O O H D317 H H D317 O O 115H FGly51 O 115H FGly51 sulfatase H sulfatase sulfatase sulfatase H H N H211 N H211 N N O O S O H Ca2+ H Ca2+ N N N N O H O O O O D317 H D317 HO HO 115H FGly51 115H FGly51 sulfatase sulfatase Sulfatase slide 100 14 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Phospholipase C slide 102 • Phospholipase C catalyses the hydrolysis of the minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, to give inositol 1,4,5-trisphosphate and diacyl glycerol, both of which are intracellular second messengers. • Phospholipase C slide 103 15 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Phospholipase C slide 104 Phospholipase C slide 105 PLC! H311 PLC! H311 H H N N Ca2+ Ca2+ N N H H H356 H B H356 H B O O O N H O N H N N O P O H O P O H O O R R R OH PLC! H311 PLC! H311 H H N N Ca2+ Ca2+ N N H H B H356 H B H356 H O O O N H O N H N N O P O H O P O O H H H O 16 Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford Phospholipase C slide 106 17 .
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