Bài giảng Biochemistry 2/e - Chapter 15: Enzyme Specificity and Regulation

Chapter 15Enzyme Specificity and Regulationto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll 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 32887-6777 Outline15.1 Specificity from Molecular Recognition15.2 Controls over Enzymatic Activity15.3 Allosteric Regulation of Enzyme Activity15.4 Allosteric Model15.5 Glycogen PhosphorylaseSPECIAL FOCUS: Hemoglobin and Myoglobin15.1 SpecificityThe 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 states15.2 Controls over Enzyme ActivitySix 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 role15.3 Allosteric RegulationAction 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")Models for Allosteric BehaviorMonod, 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 stateT state predominates in the absence of substrate SS binds much tighter to R than to TMore about MWCCooperativity is achieved because S binding increases the population of R, which increases the sites available to SLigands such as S are positive homotropic effectorsMolecules that influence the binding of something other than themselves are heterotropic effectorsGlycogen PhosphorylaseAllosteric Regulation and Covalent ModificationGP cleaves glucose units from nonreducing ends of glycogenA phosphorolysis reactionMuscle GP is a dimer of identical subunits, each with PLP covalently linkedThere is an allosteric effector site at the subunit interfaceGlycogen PhosphorylaseAllosteric Regulation and Covalent ModificationPi is a positive homotropic effectorATP is a feedback inhibitor, and a negative heterotropic effectorGlucose-6-P is a negative heterotropic effector (i.e., an inhibitor)AMP is a positive heterotrophic effector (i.e., an activator)Regulation of GP by Covalent ModificationIn 1956, Edwin Krebs and Edmond Fischer showed that a ‘converting enzyme’ could convert phosphorylase b to phosphorylase aThree years later, Krebs and Fischer show that this conversion involves covalent phosphorylationThis phosphorylation is mediated by an enzyme cascade (Figure 15.19)cAMP is a Second MessengerCyclic 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 cAMPHemoglobinA 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 146Hemoglobin 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 Hemoglobin Function Hb must bind oxygen in lungs and release it in capillaries Adjacent subunits' affinity for oxygen increases This is called positive cooperativity Myoglobin StructureMb is a monomeric heme protein Mb polypeptide "cradles" the heme group Fe in Mb is Fe2+ - 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 15.26 and discussion of CO binding The Conformation ChangeThe 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 0.029 nm - 0.29 A This change means little to Mb, but lots to Hb!Binding of Oxygen by HbThe 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!Oxygen Binding by HbA 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 0.039 nm when oxygen binds See Figure 15.32The Bohr EffectCompetition 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 Bohr Effect IICarbon dioxide diminishes oxygen binding Hydration of CO2 in tissues and extremities leads to proton production These protons are taken up by Hb as oxygen dissociates The reverse occurs in the lungs2,3-BisphosphoglycerateAn 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 effector2,3-BPG and HbThe "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