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Enzyme Specificity and Regulation Reginald Garrett and Charles Grisham. 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 - PowerPoint PPT Presentation
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Enzyme Specificity and Regulation
Reginald Garrett and Charles Grisham
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 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 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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
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
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
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 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
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 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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
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 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