Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 15 Enzyme Specificity and Regulation to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 15.1 Specificity from Molecular Recognition 15.2 Controls over Enzymatic Activity 15.3 Allosteric Regulation of Enzyme Activity 15.4 Allosteric Model 15.5 Glycogen Phosphorylase SPECIAL FOCUS : Hemoglobin and Myoglobin

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 15.1 Specificity The Result of Molecular Recognition Substrate (small) binds to enzyme (large) via weak forces - what are they? –H-bonds, van der Waals, ionic –sometimes hydrophobic interactions Understand the lock-and-key and induced-fit models Relate induced-fit to transition states

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 15.2 Controls over Enzyme Activity Six points: Rate slows as product accumulates Rate depends on substrate availability Genetic controls - induction and repression Enzymes can be modified covalently Allosteric effectors may be important Zymogens, isozymes and modulator proteins may play a role

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 15.3 Allosteric Regulation Action at "another site" Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors These effectors are usually produced elsewhere in the pathway Effectors may be feed-forward activators or feedback inhibitors Kinetics are sigmoid ("S-shaped")

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Models for Allosteric Behavior Monod, Wyman, Changeux (MWC) Model: allosteric proteins can exist in two states: R (relaxed) and T (taut) In this model, all the subunits of an oligomer must be in the same state T state predominates in the absence of substrate S S binds much tighter to R than to T

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company More about MWC Cooperativity is achieved because S binding increases the population of R, which increases the sites available to S Ligands such as S are positive homotropic effectors Molecules that influence the binding of something other than themselves are heterotropic effectors

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glycogen Phosphorylase Allosteric Regulation and Covalent Modification GP cleaves glucose units from nonreducing ends of glycogen A phosphorolysis reaction Muscle GP is a dimer of identical subunits, each with PLP covalently linked There is an allosteric effector site at the subunit interface

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glycogen Phosphorylase Allosteric Regulation and Covalent Modification P i is a positive homotropic effector ATP is a feedback inhibitor, and a negative heterotropic effector Glucose-6-P is a negative heterotropic effector (i.e., an inhibitor) AMP is a positive heterotrophic effector (i.e., an activator)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Regulation of GP by Covalent Modification In 1956, Edwin Krebs and Edmond Fischer showed that a ‘converting enzyme’ could convert phosphorylase b to phosphorylase a Three years later, Krebs and Fischer show that this conversion involves covalent phosphorylation This phosphorylation is mediated by an enzyme cascade (Figure 15.19)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company cAMP is a Second Messenger Cyclic AMP is the intracellular agent of extracellular hormones - thus a ‘second messenger’ Hormone binding stimulates a GTP- binding protein (G protein), releasing G  (GTP) Binding of G  (GTP) stimulates adenylyl cyclase to make cAMP

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Hemoglobin A classic example of allostery Hemoglobin and myoglobin are oxygen transport and storage proteins Compare the oxygen binding curves for hemoglobin and myoglobin Myoglobin is monomeric; hemoglobin is tetrameric Mb: 153 aa, 17,200 MW Hb: two alphas of 141 residues, 2 betas of 146

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Hemoglobin Function Hb must bind oxygen in lungs and release it in capillaries When a first oxygen binds to Fe in heme of Hb, the heme Fe is drawn into the plane of the porphyrin ring This initiates a series of conformational changes that are transmitted to adjacent subunits

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Hemoglobin Function Hb must bind oxygen in lungs and release it in capillaries Adjacent subunits' affinity for oxygen increases This is called positive cooperativity

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Myoglobin Structure Mb is a monomeric heme protein Mb polypeptide "cradles" the heme group Fe in Mb is Fe 2+ - ferrous iron - the form that binds oxygen Oxidation of Fe yields 3+ charge - ferric iron -metmyoglobin does not bind oxygen Oxygen binds as the sixth ligand to Fe See Figure and discussion of CO binding

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Conformation Change The secret of Mb and Hb! Oxygen binding changes the Mb conformation Without oxygen bound, Fe is out of heme plane Oxygen binding pulls the Fe into the heme plane Fe pulls its His F8 ligand along with it The F helix moves when oxygen binds Total movement of Fe is nm A This change means little to Mb, but lots to Hb!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Binding of Oxygen by Hb The Physiological Significance Hb must be able to bind oxygen in the lungs Hb must be able to release oxygen in capillaries If Hb behaved like Mb, very little oxygen would be released in capillaries - see Figure 15.22! The sigmoid, cooperative oxygen binding curve of Hb makes this possible!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Oxygen Binding by Hb A Quaternary Structure Change When deoxy-Hb crystals are exposed to oxygen, they shatter! Evidence of a structural change! One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding This massive change is induced by movement of Fe by nm when oxygen binds See Figure 15.32

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Bohr Effect Competition between oxygen and H + Discovered by Christian Bohr Binding of protons diminishes oxygen binding Binding of oxygen diminishes proton binding Important physiological significance See Figure 15.34

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Bohr Effect II Carbon dioxide diminishes oxygen binding Hydration of CO 2 in tissues and extremities leads to proton production These protons are taken up by Hb as oxygen dissociates The reverse occurs in the lungs

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 2,3-Bisphosphoglycerate An Allosteric Effector of Hemoglobin In the absence of 2,3-BPG, oxygen binding to Hb follows a rectangular hyperbola! The sigmoid binding curve is only observed in the presence of 2,3-BPG Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 2,3-BPG and Hb The "inside" story Where does 2,3-BPG bind? –" Inside" –in the central cavity What is special about 2,3-BPG? –Negative charges interact with 2 Lys, 4 His, 2 N-termini Fetal Hb - lower affinity for 2,3-BPG, higher affinity for oxygen, so it can get oxygen from mother

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company