Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 15 Enzyme Specificity and Regulation to accompany Biochemistry,

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



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



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





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















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")







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



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













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







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)









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)





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







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









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



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 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!





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 15.32









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





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





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





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









Documents

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 15 Enzyme Specificity and Regulation to accompany Biochemistry,