Andy Howard Biochemistry Lectures, Spring February 2019

Andy Howard Biochemistry Lectures, Spring 2019 21 February 2019
Enzyme Regulation Andy Howard Biochemistry Lectures, Spring February 2019

Mechanisms, Regulation, Hemoglobin
Enzymes are regulated We’ll finish our discussion of specific mechanisms, and then we’ll discuss how catalysis is regulated. Then we’ll see how hemoglobin can act as an example for many of these regulatory concepts, even though it isn’t really an enzyme. 02/21/2019 Mechanisms, Regulation, Hemoglobin

Topics for today Mechanisms: Lysozyme & TIM Regulation Thermodynamics Availability Allostery PTM Blood clotting Protein-protein Hemoglobin Structure & Dynamics Allostery Mutations 02/21/2019 Mechanisms, Regulation, Hemoglobin

Hen egg-white lysozyme
Antibacterial protectant of growing chick embryo Hydrolyzes bacterial cell-wall peptidoglycans “hydrogen atom of structural biology” Commercially available in pure form Easy to crystallize and do structure work Available in multiple crystal forms Mechanism is surprisingly complex HEWL PDB 2vb1 0.65Å 15 kDa 02/21/2019 Mechanisms, Regulation, Hemoglobin

Mechanism of lysozyme Strain-induced destabilization of substrate makes the substrate look more like the transition state Long arguments about the nature of the intermediates Accepted answer: covalent intermediate between D52 and glycosyl C1 02/21/2019 Mechanisms, Regulation, Hemoglobin

The controversy 02/21/2019 Mechanisms, Regulation, Hemoglobin

Triosephosphate isomerase (TIM)
dihydroxyacetone phosphate  glyceraldehyde-3-phosphate Km=10µM kcat=4000s-1 kcat/Km=4*108M-1s-1  DHAP Glyc-3-P 02/21/2019 Mechanisms, Regulation, Hemoglobin

TIM mechanism DHAP carbonyl H-bonds to neutral imidazole of his-95; proton moves from C1 to carboxylate of glu165 Enediolate intermediate (C—O- on C2) Unprotonated form of his95 interacts with C1—O-H; then glu165 donates proton back to C2 See Fort’s treatment CHY431/Enzyme3.html) Leishmania TIM 56 kDa dimer; monomer shown EC PDB 2VXN, 0.82Å 02/21/2019 Mechanisms, Regulation, Hemoglobin

Regulation of enzymes The very catalytic proficiency for which enzymes have evolved means that their activity must not be allowed to run amok 02/21/2019 Mechanisms, Regulation, Hemoglobin

How activity is regulated
Thermodynamics Enzyme availability Allostery Post-translational modification Protein-protein interactions 02/21/2019 Mechanisms, Regulation, Hemoglobin

Thermodynamics as a regulatory force
Remember that Go’ is not the determiner of spontaneity: G is. Thus local product and substrate concentrations determine whether the enzyme is catalyzing reversible reactions to the left or to the right Rule of thumb: Go’ < -20 kJ mol-1 is irreversible 02/21/2019 Mechanisms, Regulation, Hemoglobin

Enzyme availability The enzyme has to be where the reactants are in order for it to act Even a highly proficient enzyme has to have a nonzero concentration How can the cell control [E]tot? Transcription (and translation) Protein processing (degradation) Compartmentalization 02/21/2019 Mechanisms, Regulation, Hemoglobin

Transcriptional control
mRNAs have short lifetimes Therefore once a protein is degraded, it will be replaced and available only if new transcriptional activity for that protein occurs  Many types of transcriptional effectors 02/21/2019 Mechanisms, Regulation, Hemoglobin

Transcriptional control
Proteins can bind to their own gene Small molecules can bind to gene Promoters can be turned on or off 02/21/2019 Mechanisms, Regulation, Hemoglobin

Protein degradation All proteins have finite half-lives; Enzymes’ lifetimes often shorter than structural or transport proteins Degraded by slings & arrows of outrageous fortune; Or: activity of the proteasome, a molecular machine that tags proteins for degradation and then accomplishes it 02/21/2019 Mechanisms, Regulation, Hemoglobin

Compartmentalization
If the enzyme is in one compartment and the substrate in another, it won’t catalyze anything Many mitochondrial catabolic enzymes act on substrates produced in cytoplasm; these require elaborate transport mechanisms to move them in Therefore, control of the transporters confers control over the enzymatic system 02/21/2019 Mechanisms, Regulation, Hemoglobin

Allostery 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 Ligand may bind to same subunit or a different one These effects happen to non-enzymatic proteins as well as enzymes 02/21/2019 Mechanisms, Regulation, Hemoglobin

Substrates as allosteric effectors (homotropic)
Standard example: binding of O2 to one subunit of tetrameric hemoglobin induces conformational change that facilitates binding of 2nd (& 3rd & 4th) O2’s So the first oxygen is an allosteric effector of the activity in the other subunits Effect can be inhibitory or accelerative 02/21/2019 Mechanisms, Regulation, Hemoglobin

Other allosteric effectors (heterotropic)
Covalent modification of an enzyme by phosphate or other PTM molecules can turn it on or off Usually catabolic enzymes are stimulated by phosphorylation and anabolic enzymes are turned off, but not always Phosphatases catalyze dephosphorylation; these have the opposite effects 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Kinetics of homotropic allostery
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 1st substrate accelerates binding of 2nd and later ones 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Rigid one is typically called “tight” or “T” state; the flexible one is called “relaxed” or “R” state Allosteric effectors shift the equilibrium toward R or toward T 02/21/2019 Mechanisms, Regulation, Hemoglobin

MWC model for allostery
Emphasizes symmetry and symmetry-breaking in seeing how subunit interactions give rise to allostery Can only explain positive cooperativity 02/21/2019 Mechanisms, Regulation, Hemoglobin

Koshland, Nemethy & Filmer (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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Heterotropic effectors
02/21/2019 Mechanisms, Regulation, Hemoglobin

Aspartate transcarbamoylase
asp + carbamoyl phosphate → carbamoyl aspartate + Pi 2 catalytic trimers, & 3 regulatory dimers Allosteric sites bind ATP, CTP, UTP Binding of ATP at allosteric sites activates enzyme (T→R); UTP,CTP ↓ especially when they’re both present E.coli ATCase 305kDa oligomer EC PDB 4FYX 2.1Å 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 others external protease is involved Blood-clotting cascade involves a series of catalytic activations 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Bovine chymotrypsinogen 51 kDa homodimer EC PDB 2CGA, 1.8Å 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

2 pathways to blood clotting
Two different pathways: Intrinsic: blood sees injury directly Extrinsic: injured tissues release factors that stimulate process Come together at factor X 02/21/2019 Mechanisms, Regulation, Hemoglobin

Cascade 02/21/2019 Mechanisms, Regulation, Hemoglobin

Protein-protein interactions
One major change in biochemistry in last 30 years is increasing emphasis on protein-protein interactions in understanding biological activities Many proteins depend on exogenous partners for modulating activity up or down Example: cholera toxin’s enzymatic component depends on interaction with human protein ARF6 Cholera toxin-ARF6 complex Total MW 42.5 kDa PDB 2A5D, 1.8Å 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 reaction O2(lung)  O2 (peripheral tissues) 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Structure determinations
Photo courtesy EMBL Myoglobin & hemoglobin were the first 2 proteins to have their 3-D structures determined experimentally Myoglobin: Kendrew, 1958 Hemoglobin: Perutz, 1958 Key tool-developers Nobel prizes for both, 1965 (small T!) Photo courtesy Oregon State Library 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Iron ion in center of heme Sperm whale myoglobin; 1.4 Å 18 kDa monomer PDB 2JHO 02/21/2019 Mechanisms, Regulation, Hemoglobin

Iron coordination Hexacoordinate iron is coordinated by 4 N atoms in protoporphyrin system and by a histidine side-chain N (his F8) Sixth coordination site is occupied by O2, H2O, CO, or whatever else fits into the ligand site 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 This difference doesn’t matter much here, but it’ll matter a lot more in hemoglobin! 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Human deoxyHb 65kDa hetero-tetramer PDB 2HHB, 1.74Å 02/21/2019 Mechanisms, Regulation, Hemoglobin

Subunit interfaces in Hb
Subunit interfaces are where many allosteric interactions occur Strong interactions: 1 with 1 and 2, 1 with 1 and 2 Weaker interactions: 1 with 2, 1 with 2 Image courtesy Pittsburgh Supercomputing Center 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Relationship between subunit dynamics and monomer movements
This can be connected to the oxygen binding and the movement of the iron atom toward the heme plane 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Relaxed state for oxyHb
The stablest form for oxyhemoglobin is described as a “relaxed” or R state Accessibility of beta chains to exterior is substantially enhanced 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 oxygen to Hb stabilizes R state in the other subunits, so their affinity is higher 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Sir Archibald V. Hill photo courtesy nobelprize.org 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 dimensionless value characterizing the cooperativity n is called the Hill coefficient. 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Alternate formulation: P50n  K so Y/(1-Y) = pO2n / K 02/21/2019 Mechanisms, Regulation, Hemoglobin

Meaning of P50 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. 02/21/2019 Mechanisms, Regulation, Hemoglobin

Real Hill parameters 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 Torr So lung has Y~0.98, periphery has Y~0.06! 02/21/2019 Mechanisms, Regulation, Hemoglobin

Utility of the Hill effect
That difference (0.98 to 0.06) is big enough to be functional If n=1, Ylung=0.79, Ytissue=0.28; not nearly as good a delivery vehicle! 02/21/2019 Mechanisms, Regulation, Hemoglobin

MWC theory Monod, Wyman, Changeux developed mathematical model describing TR 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 TR equilibrium, so that tends to make more R Jacques Monod Photo Courtesy nndb.com 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Dan Koshland Photo Courtesy U. of California 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 Christian Bohr photo courtesy Wikipedia 02/21/2019 Mechanisms, Regulation, Hemoglobin

How the Bohr effect happens
R form has an effective pKa that is lower than T One reason: In T state, his146 is close to asp 94. Thus histidine pKa is higher In R state, his146 is farther from asp 94 so its pKa is lower. Cartoon courtesy Jon Robertus, UT Austin 02/21/2019 Mechanisms, Regulation, Hemoglobin

Physiological result of Bohr effect
Actively metabolizing tissues tend to produce lower pH That promotes O2 release where it’s needed 02/21/2019 Mechanisms, Regulation, Hemoglobin

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- Bicarbonate transported back to lungs When Hb gets re-oxygenated, bicarbonate gets converted back to gaseous CO2 and exhaled 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Another allosteric effector
2,3-bisphosphoglycerate: heterotropic allosteric effector 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! 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Fetal hemoglobin Higher oxygen affinity because the type of hemoglobin found there has a lower affinity for BPG Fetal Hb is 22;  doesn’t bind BPG as much as . That helps ensure that plenty of O2 gets from mother to fetus across the placenta 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Why has this mutation survived?
Homozygotes don’t generally survive to produce progeny; but heterozygotes do Heterozygotes do have modestly reduced oxygen-carrying capacity in their blood because some erythrocytes are sickled Deoxy HbS 2.05 Å PDB 2HBS 02/21/2019 Mechanisms, Regulation, Hemoglobin

Heterozygotes and malaria
BUT heterozygotes are somewhat resistant to malaria, so the gene survives in tropical places where malaria is a severe problem 02/21/2019 Mechanisms, Regulation, Hemoglobin

How is sickling related to malaria?
Malaria parasite (Plasmodium 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 Plasmodium falciparum from A.Dove (2001) Nature Medicine 7:389 02/21/2019 Mechanisms, Regulation, Hemoglobin

Malaria and the tropics
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) 02/21/2019 Mechanisms, Regulation, Hemoglobin

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 02/21/2019 Mechanisms, Regulation, Hemoglobin

Andy Howard Biochemistry Lectures, Spring February 2019

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Andy Howard Biochemistry Lectures, Spring February 2019

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