Enzyme Regulation

Enzyme Regulation Andy HowardIntroductory Biochemistry 12 November 2008 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Mechanism Topics Biochem: Enzyme Regulation

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) Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

MWC model for allostery • Emphasizes symmetry and symmetry-breaking in seeing how subunit interactions give rise to allostery • Can only explain positive cooperativity Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

Heterotropic effectors Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

Cascade Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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) Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

Structure determinations Photo courtesyEMBL • 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!) Photo courtesyOregon State Library Biochem: Enzyme Regulation

Myoglobin structure Sperm whale myoglobin; 1.4 Å18 kDa monomerPDB 2JHO • 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 Biochem: Enzyme Regulation

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! Biochem: Enzyme Regulation

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 deoxyHbPDB 2HHB1.74Å65kDa hetero-tetramer Biochem: Enzyme Regulation

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 Image courtesy PittsburghSupercomputingCenter Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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. Biochem: Enzyme Regulation

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 soY/(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! Biochem: Enzyme Regulation

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! Biochem: Enzyme Regulation

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 MonodPhoto Courtesy Nobelprize.org Biochem: Enzyme Regulation

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 KoshlandPhoto Courtesy U. of California Biochem: Enzyme Regulation

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) Christian Bohrphoto courtesyWikipedia Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

Physiological result of Bohr effect • Actively metabolizing tissues tend to produce lower pH • That promotes O2 release where it’s needed Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

Another allosteric effector BPG (Wikimedia) • 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! Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

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 Deoxy HbS2.05 ÅPDB 2HBS Biochem: Enzyme Regulation

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) Plasmodium falciparumfrom A.Dove (2001) Nature Medicine 7:389 Biochem: Enzyme Regulation

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 Biochem: Enzyme Regulation

Enzyme Regulation