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Chapter 15

Chapter 15. Enzyme Specificity and Regulation to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham.

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Chapter 15

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  1. 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 32887-6777

  2. 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

  3. 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

  4. 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

  5. 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")

  6. 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

  7. 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

  8. Glycogen PhosphorylaseAllosteric 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

  9. Glycogen PhosphorylaseAllosteric Regulation and Covalent Modification • Pi 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)

  10. 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)

  11. 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

  12. 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

  13. Hemoglobin FunctionHb 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

  14. Hemoglobin FunctionHb must bind oxygen in lungs and release it in capillaries • Adjacent subunits' affinity for oxygen increases • This is called positive cooperativity

  15. Myoglobin Structure Mb 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 - ferriciron -metmyoglobin does not bind oxygen • Oxygen binds as the sixth ligand to Fe • See Figure 15.26 and discussion of CO binding

  16. 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 0.029 nm - 0.29 A • This change means little to Mb, but lots to Hb!

  17. 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!

  18. 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 0.039 nm when oxygen binds • See Figure 15.32

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