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Enzymes: A Molecular Perspective Shuchismita Dutta, Ph.D. Learning Objectives • Introduction to Enzymes • Enzyme action • Enzyme regulation Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Learning Objectives • Introduction to Enzymes • Enzyme action • Enzyme regulation Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Enzymes Structure and Composition • Most enzymes are proteins • Some enzymes are RNAs • Enzyme function (catalysis) is facilitated by specific amino acids or groups in the molecule along with ions, cofactors etc. Functions • Catalyzes specific reaction • Reusable molecule Substrate(s) + Enz Product(s) + Enz Starch Amylase Salivary and Pancreatic Amylase enzymes digest starch http://pdb101.rcsb.org/motm/74 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Enzymes Are Chemical Catalysts • Speed up Reaction rate • Sometimes by million fold • Lower energy barrier for Reaction – Temporarily stabilizes interaction between substrates – Forms Transition state complexes – Helps form Product(s) http://www.ncbi.nlm.nih.gov/books/NBK9921/figure/A281/ Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Amylase Digests Starch Amylase (enzyme) Site of substrate cleavage Zn Cl Part of starch molecule (substrate) http://pdb101.rcsb.org/motm/74 Active site residues Glu 233, Asp 197, Asp 300 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Learning Objectives • Introduction to Enzymes • Enzyme action • Enzyme regulation Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Enzyme Action Models Lock and Key • Emil Fischer (1894) • Active site rigid, substrate(s) fit(s) precisely into enzyme active site Lock and Key Model Induced fit • Daniel Koshland (1959) • Binding of substrate(s) changes conformations of substrate(s) and enzyme Induced Fit http://www.ncbi.nlm.nih.gov/books/NBK9921/figure/A284/ Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Hexokinase: Induced Fit Model • Hexokinase (HK) transfers a phosphate from ATP to glucose (forms glucose 6phosphate) • Catalyzes first step in glycolysis • Shaped like a clamp - big groove in one side • HK structure without glucose (open); with glucose (closed) Hexokinase without glucose (open) Hexokinase with glucose (closed) http://pdb101.rcsb.org/motm/50 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Catalytic Mechanism • Residues in catalytic site conserved in enzyme families (e.g. Serine Proteases) • Residues for peptide bond cleavage in all Serine Protease enzymes – Serine – Histidine – Aspartate Trypsin Chymotrypsin http://pdb101.rcsb.org/motm/46 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Elastase Substrate Specificity Property Trypsin Chymotrypsin Elastase Enzyme cuts after positive amino acids bulky amino acids small uncharged amino acids Substrate has Lysine, Arginine Phenylalanine, Tryptophan Alanine Enzyme specificity pocket has Aspartate Serine Valine, Threonine Active site: Histidine Serine Specificity pocket residues PDB ID:2ptn PDB ID:2cha Developed as part of the RCSB Collaborative Curriculum Development Program 2016 PDB ID:3est Coenzymes • Work together with enzyme • Small molecules, bound to enzymes to play critical roles • May act as carrier of specific atoms or groups • Are altered during enzyme reactions • Are recycled and may take part in many different reactions. – Nicotinamide adenine dinucleotide (NAD+) • carries electrons in oxidationreduction reactions • can accept a hydrogen ion (H+) and two electrons (e-) to form NADH • NADH can donate electrons to a second substrate, re-forming NAD+ Glyceraldehyde-3-Phosphate Dehydrogenase active site with bound NAD+ http://pdb101.rcsb.org/motm/5 0 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Learning Objectives • Introduction to Enzymes • Enzyme action • Enzyme regulation Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Allosteric Regulation • Binding of a small molecule outside the catalytic site • Regulator binding alters overall shape and interactions of enzyme • Binding may specifically activate or inhibit enzyme activity http://www.ncbi.nlm.nih.gov/books/NBK9921/figure/A292 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Pyruvate Kinase: Allosteric Activation • Pyruvate kinase makes ATP only when needed • Composed of four flexible subunits arranged as a diamond shape • Inhibited by ATP and amino acids • Activated by Fructose-1,6Bisphosphate into flexed shape. Inactive Enzyme Active Enzyme Catlytic Sites Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Regulatory Sites Enzyme Inhibition • Why? – To prevent excessive function within cells/organism • How? Trypsinogen: has extra piece of protein chain to cover active site and keep it inactive Pancreatic Trypsin Inhibitor ensures that any traces of active Trypsin are inactivated when not necessary – Portion of Enzyme or another protein blocks access to enzyme active site http://pdb101.rcsb.org/motm/46 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Enzyme Inhibitors as Drugs • HIV-1 Protease bound to substrate peptide • HIV Protease inhibitors as Drugs: Substrate-like or other molecules bind to protease active site block HIV-1 protease function (viral maturation) Indinavir Saquinavir Ritonavir Nelfinavir http://pdb101.rcsb.org/motm/6 Developed as part of the RCSB Collaborative Curriculum Development Program 2016 Summary • Introduction to Enzymes – Enzymes and Catalysis • Enzyme action – Enzyme Action models – Catalytic mechanisms, specificity and coenzymes • Enzyme regulation – Allosteric Regulation – Inhibition and Drugs Developed as part of the RCSB Collaborative Curriculum Development Program 2016