11/12/2009Biochem: Enzyme Regulation Enzyme Regulation Andy Howard Introductory Biochemistry 12...
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Transcript of 11/12/2009Biochem: Enzyme Regulation Enzyme Regulation Andy Howard Introductory Biochemistry 12...
11/12/2009Biochem: Enzyme Regulation
Enzyme Regulation
Andy HowardIntroductory Biochemistry
12 November 2008
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Enzymes are under several levels of control
Some controls operate at the level of enzyme availability
Other controls are exerted by thermodynamics, inhibition, or allostery
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Mechanism Topics
Globins as Examples Oxygen binding Tertiary structure
Quarternary structure
R and T states Allostery Bohr effect BPG as an effector
Sickle-cell anemia
Allostery:an example
Post-translational modification
Protein-protein interactions
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Allostery (review)
Remember we defined this as an effect on protein activity in which binding of a ligand to a protein induces a conformational change that modifies the protein’s activity
Ligand may be the same molecule as the substrate or it may be a different one (homotropic vs. heterotropic)
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Cyclic AMP-dependent protein kinases
Enzymes phosphorylate proteins with S or T within sequence R(R/K)X(S*/T*)
Intrasteric control:regulatory subunit or domain has a sequence that looks like the target sequence; this binds and inactivates the kinase’s catalytic subunit
When regulatory subunits binds cAMP, it releases from the catalytic subunit so it can do its thing
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Kinetics of allosteric enzymes Generally these don’t obey Michaelis-Menten kinetics
Homotropic positive effectors produce sigmoidal (S-shaped) kinetics curves rather than hyperbolae
This reflects the fact that the binding of the first substrate accelerates binding of second and later ones
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T R State transitions Many allosteric effectors influence the equilibrium between two conformations
One is typically more rigid and inactive, the other is more flexible and active
The rigid one is typically called the “tight” or “T” state; the flexible one is called the “relaxed” or “R” state
Allosteric effectors shift the equilibrium toward R or toward T
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MWC model for allostery Emphasizes
symmetry and symmetry-breaking in seeing how subunit interactions give rise to allostery
Can only explain positive cooperativity
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Koshland (KNF) model Emphasizes conformational changes from one state to another, induced by binding of effector
Ligand binding and conformational transitions are distinct steps
… so this is a sequential model for allosteric transitions
Allows for negative cooperativity as well as positive cooperativity
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Heterotropic effectors
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Post-translational modification We’ve already looked at phosphorylation
Proteolytic cleavage of the enzyme to activate it is another common PTM mode
Some proteases cleave themselves (auto-catalysis); in other cases there’s an external protease involved
Blood-clotting cascade involves a series of catalytic activations
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Zymogens As mentioned earlier, this is a term for an inactive form of a protein produced at the ribosome
Proteolytic post-translational processing required for the zymogen to be converted to its active form
Cleavage may happen intracellularly, during secretion, or extracellularly
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Blood clotting
Seven serine proteases in cascade Final one (thrombin) converts fibrinogen to fibrin, which can aggregate to form an insoluble mat to prevent leakage
Two different pathways: Intrinsic: blood sees injury directly Extrinsic: injured tissues release factors that stimulate process
Come together at factor X
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Cascade
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Protein-protein interactions One major change in biochemistry in the last 20 years is the increasing emphasis on protein-protein interactions in understanding biological activities
Many proteins depend on exogenous partners for modulating their activity up or down
Example: cholera toxin’s enzymatic component depends on interaction with human protein ARF6
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Globins as aids to understanding Myoglobin and hemoglobin are well-understood non-enzymatic proteins whose properties help us understand enzyme regulation
Hemoglobin is described as an “honorary enzyme” in that it “catalyzes” the reactionO2(lung) O2 (peripheral tissues)
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Setting the stage for this story
Myoglobin is a 16kDa monomeric O2-storage protein found in peripheral tissues
Has Fe-containing prosthetic group called heme; iron must be in Fe2+ state to bind O2
It yields up dioxygen to various oxygen-requiring processes, particularly oxidative phosphorylation in mitochondria in rapidly metabolizing tissues
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Why is myoglobin needed? Free heme will bind O2 nicely;why not just rely on that?
Protein has 3 functions: Immobilizes the heme group Discourages oxidation of Fe2+ to Fe3+
Provides a pocket that oxygen can fit into
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Setting the stage II Hemoglobin (in vertebrates, at least) is a tetrameric, 64 kDa transport protein that carries oxygen from the lungs to peripheral tissues
It also transports acidic CO2 the opposite direction
Its allosteric properties are what we’ll discuss
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Structure determinations Myoglobin & hemoglobin were the
first 2 proteins to have their 3-D structures determined experimentally Myoglobin: Kendrew, 1958 Hemoglobin: Perutz, 1958 Most of the experimental tools that crystallographers rely on were developed for these structure determinations
Nobel prizes for both, 1965 (small T!)
QuickTime™ and a decompressor
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Photo courtesyOregon State Library
Photo courtesyEMBL
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Myoglobin structure
Almost entirely -helical 8 helices, 7-26 residues each Bends between helices generally short Heme (ferroprotoporphyrin IX) tightly but noncovalently bound in cleft between helices E&F
Hexacoordinate iron is coordinated by 4 N atoms in protoporphyrin system and by a histidine side-chain N (his F8): fig.15.25
Sixth coordination site is occupied by O2, H2O, CO, or whatever else fits into the ligand site
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Sperm whale myoglobin; 1.4
Å18 kDa monomer
PDB 2JHO
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O2 binding alters myoglobin structure a little
Deoxymyoglobin: Fe2+ is 0.55Å out of the heme plane, toward his F8, away from O2 binding site
Oxymyoglobin: moves toward heme plane—now only 0.26Å away (fig.15.26)
This difference doesn’t matter much here, but it’ll matter a lot more in hemoglobin!
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Hemoglobin structure
Four subunits, each closely resembling myoglobin in structure (less closely in sequence);H helix is shorter than in Mb
2 alpha chains,2 beta chains
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Human deoxyHbPDB 2HHB1.74Å65kDa hetero-tetramer
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Subunit interfaces in Hb
Subunit interfaces are where many of the allosteric interactions occur Strong interactions:
1 with 1 and 2,1 with 1 and 2
Weaker interactions:1 with 2, 1 with 2
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Image courtesyPittsburghSupercomputingCenter
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Subunit dynamics 1-1 and 2-2 interfaces are solid and don’t change much upon O2 binding
1-2 and 2-1 change much more:the subunits slide past one another by 15º Maximum movement of any one atom ~ 6Å Residues involved in sliding contacts are in helices C, G, H, and the G-H corner
This can be connected to the oxygen binding and the movement of the iron atom toward the heme plane
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Conformational states
We can describe this shift as a transition from one conformational state to another
The stablest form for deoxyHb is described as a “tense” or T state Heme environment of beta chains is almost inaccessible because of steric hindrance
That makes O2 binding difficult to achieve The stablest form for oxyHB is described as a “relaxed” or R state
Accessibility of beta chains substantially enhanced
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Hemoglobin allostery Known since early 1900’s that hemoglobin displayed sigmoidal oxygen-binding kinetics
Understood now to be a function of higher affinity in 2nd, 3rd, 4th chains for oxygen than was found in first chain
This is classic homotropic allostery even though this isn’t really an enzyme
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R T states and hemoglobin
We visualize each Hb monomer as existing in either T (tight) or R (relaxed) states; T binds oxygen reluctantly, R binds it enthusiastically
DeoxyHb is stablest in T state Binding of first Hb stabilizes R state in the other subunits, so their affinity is higher
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Binding and pO2
Hill found that that binding could be modeled by a polynomial fit to pO2
Kinetics worked out in 1910’s: didn’t require protein purification, just careful in vitro measurements of blood extracts
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Sir Archibald V. Hill photo courtesy nobelprize.org
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Hill coefficients Actual equation is on next page Relevant parameters to determine are P50, the oxygen partial pressure at which half the O2-binding sites are filled, and n, a unitless value characterizing the cooperativity
n is called the Hill coefficient.
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pO2 and fraction oxygenated
If Y is fraction of globin that is oxygenated and pO2 is the partial pressure of oxygen,then Y/(1-Y) = (pO2 /P50)n
4th-edition formulation: P50n K so
Y/(1-Y) = pO2n / K
P50 is a parameter corresponding to half-occupied hemoglobin work out the algebra: When pO2 = P50, Y/(1-Y) = 1n=1 so Y = 1/2.
Note that the equation on p.496 of the enhanced 3rd edition is wrong!
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Real Hill parameters (p.496)
Human hemoglobin has n ~ 2.8, P50 ~ 26 Torr Perfect cooperativity, tetrameric protein: n =4
No cooperativity at all would be n = 1. Lung pO2 ~ 100 Torr;peripheral tissue 10-40 Torr
So lung has Y~0.98, periphery has Y~0.06! That’s a big enough difference to be functional
If n=1, Ylung=0.79, Ytissue=0.28; not nearly as good a delivery vehicle!
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MWC theory Monod, Wyman, Changeux developed mathematical model describing TR transitions and applied it to Hb
Accounts reasonably well for sigmoidal kinetics and Hill coefficient values
Key assumption:ligand binds only to R state,so when it binds, it depletes R in the TR equilibrium,so that tends to make more R
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Jacques MonodPhoto Courtesy Nobelprize.org
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Koshland’s contribution
Conformational changes between the two states are also clearly relevant to the discussion
His papers from the 1970’s articulating the algebra of hemoglobin-binding kinetics are amazingly intricate
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Dan KoshlandPhoto Courtesy U. of California
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Added complication I: pH Oxygen affinity is pH dependent
That’s typical of proteins, especially those in which histidine is involved in the activity (remember it readily undergoes protonation and deprotonation near neutral pH)
Bohr effect (also discovered in early 1900’s): lower affinity at low pH (fig. 15.33)
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Christian Bohrphoto courtesyWikipedia
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How the Bohr effect happens
R form has an effective pKa that is lower than T
One reason: In the T state, his146 is close to asp 94. That allows the histidine pKa to be higher
In R state, his146 is farther from asp 94 so its pKa is lower.
Cartoon courtesy Jon Robertus, UT Austin
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Physiological result of Bohr effect Actively metabolizing tissues tend to produce lower pH
That promotes O2 release where it’s needed
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CO2 also promotes dissociation High [CO2] lowers pH because it dissolves with the help of the enzyme carbonic anhydrase and dissociates:H2O + CO2 H2CO3 H+ + HCO3
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Bicarbonate transported back to lungs When Hb gets re-oxygenated, bicarbonate gets converted back to gaseous CO2 and exhaled
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Role of carbamate
Free amine groups in Hb react reversibly with CO2 to form R—NH—COO- + H+
The negative charge on the amino terminus allows it to salt-bridge to Arg 141
This stabilizes the T (deoxy) state
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Another allosteric effector 2,3-bisphosphoglycerate is a heterotropic allosteric effector of oxygen binding
Fairly prevalent in erythrocytes (4.5 mM); roughly equal to [Hb]
Hb tetramer has one BPG binding site BPG effectively crosslinks the 2 chains
It only fits in T (deoxy) form!
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BPG (Wikimedia)
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BPG and physiology
pO2 is too high (40 Torr) for efficient release of O2 in many cells in absence of BPG
With BPG around, T-state is stabilized enough to facilitate O2 release
Big animals (e.g. sheep) have lower O2 affinity but their Hb is less influenced by BPG
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Fetal hemoglobin
Higher oxygen affinity because the type of hemoglobin found there has a lower affinity for BPG
Fetal Hb is 22; doesn’t bind BPG as much as .
That helps ensure that plenty of O2
gets from mother to fetus across the placenta
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Sickle-cell anemia
Genetic disorder: Hb residue 6 mutated from glu to val. This variant is called HbS.
Results in intermolecular interaction between neighboring Hb tetramers that can cause chainlike polymerization
Polymerized hemoglobin will partially fall out of solution and tug on the erythrocyte structure, resulting in misshapen (sickle-shaped) cells
Oxygen affinity is lower because of insolubility
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Why has this mutation survived?
Homozygotes don’t generallysurvive to produce progeny;but heterozygotes do
Heterozygotes do have modestly reduced oxygen-carrying capacity in their blood because some erythrocytes are sickled
BUT heterozygotes are somewhat resistant to malaria, so the gene survives in tropical places where malaria is a severe problem
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Deoxy HbS2.05 Å
PDB 2HBS
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How is sickling related to malaria? Malaria parasite (Plasmondium spp.) infects erythrocytes
They’re unable to infect sickled cells
So a partially affected cell might survive the infection better than a non-sickled cell
Still some argument about all of this
Note that most tropical environments have plenty of oxygen around (not a lot of malaria at 2000 meters elevation)
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Plasmodium falciparumfrom A.Dove (2001) Nature Medicine 7:389
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Other hemoglobin mutants Because it’s easy to get human blood, dozens of hemoglobin mutants have been characterized
Many are asymptomatic Some have moderate to severe effects on oxygen carrying capacity or erythrocyte physiology