biochemistry 2/e - garrett & grisham copyright © 1999 by harcourt brace & company chapter...
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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 32887-6777
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 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 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 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")
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
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Biochemistry 2/e - Garrett & Grisham
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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
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 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)
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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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
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Biochemistry 2/e - Garrett & Grisham
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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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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HemoglobinA 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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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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 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
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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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!
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 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!
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
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Biochemistry 2/e - Garrett & Grisham
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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 15.32
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
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Biochemistry 2/e - Garrett & Grisham
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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 CO2 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-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 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 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
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