Biochem_2023_Ch 4 pt2.pdf

Section 4.3 The Mechanism of an Enzyme Can Be Deduced from Structural, Kinetic, and Spectral Data Copyright © 2019 John Wiley & Sons, Inc. General Principles of Enzyme Catalyzed Reactions ▪ Enzymes bind to substrates using weak forces and orient them for chemistry to occur. → Entropy is lowered....

Section 4.3 The Mechanism of an Enzyme Can Be Deduced from Structural, Kinetic, and Spectral Data Copyright © 2019 John Wiley & Sons, Inc. General Principles of Enzyme Catalyzed Reactions ▪ Enzymes bind to substrates using weak forces and orient them for chemistry to occur. → Entropy is lowered. ▪ Enzymes bind substrates using a large number of weak forces. This is sometimes referred to as electrostatic catalysis and is the sum total of the weak forces acting on the substrate to effect chemical change. ▪ In induced fit, the enzymes bind substrates that favor the transition state. Copyright © 2019 John Wiley & Sons, Inc. Types of Catalysis ▪ General acid–base catalysis • Amino acid side chains donate or accept protons. • Polar and charged amino acids play important roles. Copyright © 2019 John Wiley & Sons, Inc. Types of Catalysis ▪ Metal ion catalysis • Active site metal can act as a redox active center. • Metal ions are found in a third of all enzymes. Copyright © 2019 John Wiley & Sons, Inc. Types of Catalysis ▪ Covalent catalysis • Nucleophilic or electrophilic attack on an atom results in a covalent intermediate. • Involves Ser, Asp, Cys, Lys, Tyr, and several cofactors Copyright © 2019 John Wiley & Sons, Inc. Lysozyme: An Example of Acid–Base Catalysis ▪ A natural antibiotic ▪ An enzyme found in tears and mucus ▪ A hydrolase that cleaves a link between carbohydrate chains found in the cell wall of bacteria. ▪ The active site contains two negatively charge residues ▪ Glutamic acid is protonated, whereas aspartate is not Copyright © 2019 John Wiley & Sons, Inc. Anhydrase: An Example of Metal Ion Catalysis ▪ Found in erythrocytes. ▪ Catalyzes the conversion of carbon dioxide to carbonic acid. ▪ Zn2+ is in the active site. Figure 4.14C Carbonic anhydrase structure and reaction. Copyright © 2019 John Wiley & Sons, Inc. Mechanism of Anhydrase Figure 4.15 Mechanism of carbonic anhydrase. Copyright © 2019 John Wiley & Sons, Inc. Proteases Defined ▪ Proteases are enzymes that degrade proteins. ▪ Many proteases have a Trypsin fold = structure comprised of two β barrel domains, the active site being at the interface of the two. • Cleave peptide bonds • Some are specific. o Ser, Asp, and Cys proteases, metalloproteases • Important in protein maturation, blood clotting, and protein trafficking • Include chymotrypsin, trypsin, and elastase Copyright © 2019 John Wiley & Sons, Inc. Protease Examples Serine proteases, aspartyl proteases, metalloproteases, and cysteine proteases all employ similar chemistry: nucleophilic attack on the carbonyl carbon. Note that these enzymes not only employ similar nucleophiles, they have a conserved topology and overall structure, although not necessarily the same amino acid sequence. Figure 4.18 Different classes of proteases. Copyright © 2019 John Wiley & Sons, Inc. Chymotrypsin: An Example of Covalent Catalysis ▪ Serine protease ▪ Cleaves dietary protein on the C-terminus of Tyr, Phe, and Trp ▪ Secreted from the pancreas ▪ Contains a catalytic triad containing Aspartate, Histidine, and Serine ▪ Inactive form = chymotrypsinogen Copyright © 2019 John Wiley & Sons, Inc. Chymotrypsin Structure and Reaction Figure 4.16 Chymotrypsin structure and reaction. Copyright © 2019 John Wiley & Sons, Inc. Chymotrypsin Mechanism ▪ Nucleophilic attack Figure 4.17 Mechanism of chymotrypsin. Copyright © 2019 John Wiley & Sons, Inc. Affinity Labeling Defined ▪ Affinity labeling is a technique used to specifically label residues on the active site. ▪ Examples include: • Diisopropylfluroidate (DIPF)—serine active site • Tosyl-L-phenylalanine chloromethyl ketone (TPCK) —histidine active site ▪ Labeling can be blocked by competitive inhibitors. Copyright © 2019 John Wiley & Sons, Inc. Affinity Labels Figure 4.19 Affinity labels diisofluorophosphate (DIFP) and tosyl-Lphenylalanine ketone (TPCK) Copyright © 2019 John Wiley & Sons, Inc. Section 4.4 Examples of Enzyme Regulation ▪ Describe several examples that illustrate the diversity of protein structures and functions. Copyright © 2019 John Wiley & Sons, Inc. Enzyme Regulation ▪ Activity can be regulated by altering gene expression ▪ Sequestration of the enzyme in one compartment of the cells or one organ ▪ Limit the access of the enzyme to the substrate ▪ Methods include: • Covalent modification and allosteric regulation Copyright © 2019 John Wiley & Sons, Inc. Covalent Modification Defined ▪ Covalent modification is the covalent addition or removal of groups from proteins. ▪ Two common means of activation are proteolytic cleavage and phosphorylation. Copyright © 2019 John Wiley & Sons, Inc. Zymogens Defined ▪ Zymogens are inactive enzyme precursors that require proteolytic activation. ▪ Chymotrypsinogen is the inactive form of chymotrypsin. Copyright © 2019 John Wiley & Sons, Inc. Proteolytic Cleavage Defined ▪ Proteolytic cleavage is an example of enzyme activation. ▪ An inactive enzyme becomes active through cleavage. From: https://www.youtube.com/watch?v=DFngh7ow3Cw Copyright © 2019 John Wiley & Sons, Inc. Phosphorylation ▪ Another example of protein activation ▪ Facilitated by protein kinases ▪ Protein kinases add phosphate groups to the hydroxyl groups of serine, threonine, or tyrosine (from ATP donor) ▪ Phosphatases remove phosphate groups ▪ The regulation of kinases and phosphatases occurs through a series of signaling steps termed a signaling cascade. Copyright © 2019 John Wiley & Sons, Inc. Src: An Example of Regulation via Phosphorylation Figure 4.22 Src is regulated by phosphorylation. Copyright © 2019 John Wiley & Sons, Inc. Allosteric Regulation ▪ Increases or decreases the enzymatic activity by binding at a site other than the active site ▪ Most rapid and most direct form of regulation ▪ Two states: Relaxed (R) and Tense (T) Figure 4.23 Allosteric regulation. Copyright © 2019 John Wiley & Sons, Inc. Allosteric Regulation Binding Curves ▪ Exhibit sigmoidal activity curves Figure 4.24 Allosteric enzymes have sigmoidal kinetics. Copyright © 2019 John Wiley & Sons, Inc. Copyright Copyright © 2019 John Wiley & Sons, Inc. All rights reserved. Reproduction or translation of this work beyond that permitted in Section 117 of the 1976 United States Act without the express written permission of the copyright owner is unlawful. Request for further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. The purchaser may make back-up copies for his/her own use only and not for distribution or resale. The Publisher assumes no responsibility for errors, omissions, or damages, caused by the use of these programs or from the use of the information contained herein. Copyright © 2019 John Wiley & Sons, Inc.