Chapter 15 -- Enzyme Regulation

What is an isozyme?

An isozyme is different "forms" of the same enzyme (structurally different, functionally the same). LDH (lactate dehydrogenase) is a tetramer (4 subunits) and is an isozyme that can exist in 5 different forms. Each 'form' catalyzes at a rate different than the other forms.

Why are allosteric enzymes oligomeric (more than one subunit)?

As a rule, allosteric enzymes are oligomeric, with each monomer possessing a substrate binding site and an allosteric site where effectors bind.

What happens to the rate of an enzymatic reaction as product accumulates?

As product accumulates, the apparent rate of the enzymatic reaction will decrease.

What do intersubunit interactions lead to?

Intersubunit interactions can lead to conformational transitions that make it easier (or harder) for additional equivalents of ligand (S, A, or I) to bind to the enzyme. S = substrate A = activator I = inhibitor

What atom is involved in heme?

Iron 2+ is in the center of the structure.

What does the kinetics curve look like for allosteric regulation?

Kinetics are sigmoid (S-shaped) (Top hyperbolic plot = MM kinetics; bottom sigmoidal plot - allosteric regulation)

What are homotropic effectors?

Ligands such as S are positive homotropic effectors. (S binding makes it more likely that more S will bind due to the increase in R as S increases.)

What does the MWC (symmetry) model tell us?

MWC model = symmetry model (each of the subunits in an allosteric enzyme are symmetric) - 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 (symmetric) - T state predominates in the absence of substrate S - S binds much tighter to R than to T

What are heterotropic effectors?

Molecules that influence the binding of something other than themselves are heterotropic effectors. A = positive heterotropic effector; its binding to E makes it more likely that S will bind to E I = negative heterotropic effector; I binding to E makes it less likely for S to bind to E

Where does 2,3-BPG bind?

"Inside" the central cavity of Hb.

Explain how glycogen phosphorylase is regulated by covalent modification.

- A "converting enzyme" can convert phosphorylase b to phosphorylase a involving covalent phosphorylation - The "converting enzyme" is named phosphorylase kinase - This phosphorylation is mediated by an enzyme cascade

What are allosteric effectors?

- Allosteric = action at "another site" - Enzymes situated at key steps in metabolic pathways are regulated by allosteric effectors. - These effectors are usually produced elsewhere in the pathway. - Effectors may be feed-forward activators (a later reaction) or feedback (an earlier reaction) inhibitors

Explain how H+ presence affects the binding of O2 by Hb.

- Deoxy-Hb has a higher affinity for H+ than oxy-Hb - Thus, as pH decreases, dissociation of O2 from Hb is enhanced

How can enzyme activity be regulated?

- Enzyme activity can be regulated allosterically - Enzyme activity can be regulated through covalent modification - Zymogens, isozymes, and modulator proteins may play a role

What amino acid changes in the alpha/gamma chains allow O2 to bind more tightly?

- Fetal gamma-chains have a Ser instead of a His at position 143 and thus lack two of the positive charges in the BPG-binding cavity - BPG binds less tightly, and Hb F thus looks more like Mb (MM kinetics) in its O2-binding behavior

What amino acid change causes sickle cell anemia?

- Glu at position 6 of the beta-chains is replaced by Val - As a result, Hb S molecules aggregate into long, chainlike polymeric structures

What is glycogen phosphorylase, and what is its function?

- Glycogen phosphorylase (GP) is an example of the many enzymes that are regulated both by allosteric controls and by covalent modification - GP cleaves glucose units from nonreducing ends of glycogen - This converts glycogen into readily usable fuel in the form of glucose-1-phosphate, a phosphorolysis reaction

What is the structure of glycogen phosphorylase?

- Glycogen phosphorylase is a dimer of identical 842 residue subunits - Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface - A regulatory phosphorylation site is located at Ser on each subunit - An allosteric effector site exerts regulatory control

Explain how CO2 promotes the dissociation of O2 from Hb.

- Hydration of CO2 in tissues and extremities leads to H+ production - These protons are taken up by Hb as oxygen dissociates - The presence of CO2has the same effect as having the presence of H+

Explain how O2 binding alters Mb conformation.

- In deoxymyoglobin, the ferrous ion lies above the plane of the heme - When O2 binds to Fe in heme of Mb, the heme Fe is drawn toward the plane of the porphyrin ring - With O2 bound, the Fe2+ atom is only slightly above the plane - This small change has little importance, however, in Hb, it is a much larger change in conformation.

Differentiate between the MWC and the KNF models of allosteric regulation.

- In the MWC model, the different conformations have different affinities for the various ligands, and the concept of ligand-induced conformational changes is ignored (all about equilibrium) - In contrast, the KNF model is based on ligand-induced conformational changes

Explain how 2,3-BPG acts as an allosteric effector of Hb.

- In the absence of 2,3-BPG, O2 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 O2 binds, it is acting as an allosteric effector

What kind of kinetics does muscle glycogen phosphorylase exhibit?

- MWC model (allosteric regulation) - Muscle glycogen phosphorylase shows cooperativity in substrate binding

Enzyme regulation by reversible covalent modification:

- Phosphate group comes from ATP in the ATP --> ADP rxn - Protein kinase is the enzyme that phosphorylates Kinase = add a phosphate Phosphatase = remove a phosphate *phosphorylating an enzyme does not always make it inactive; depends on the enzyme*

How is phosphorylation accomplished?

- Phosphorylation is accomplished via protein kinases - Each protein kinase targets specific proteins for phosphorylation

Where on proteins are protein kinases able to phosphorylate?

- Protein kinases phosphorylate Ser, Thr, and Tyr residues in target proteins (all have -OH groups in R groups) - kinases typically recognize specific amino acid sequences in their targets - Despite this specificity, all kinases share a common catalytic mechanism based on a conserved core kinase domain of about 260 residues

Describe other modes of covalent modification.

- Several hundred different chemical modifications of proteins have been discovered - Only a few of these are used to achieve metabolic regulation through reversible conversion of an enzyme between active and inactive forms - Three of the modifications (in the table) require nucleoside triphosphates (ATP, UTP) that are related to cellular energy status

What does the KNF (sequential) model tell us?

- The KNF model relies on the idea that ligand binding triggers a conformation change in a protein (K = conformation) - If the protein is oligomeric, ligand-induced conformation changes in one subunit may lead to conformation changes in adjacent subunits - The KNF model explains how ligand-induced conformation changes could cause subunits to adopt conformations with little affinity for the ligand (negative cooperativity)

Explain why fetal Hb has a higher affinity for O2 because it has lower affinity for 2,3-BPG.

- The fetus depends on its mother for O2, but its circulatory system is entirely independent - Gas exchange takes place across the placenta - Fetal Hb differs from adult Hb-- with gamma-chains in place of beta-chains,a nd thus an alpha2gamma2 structure - As a result, fetal Hb has a higher affinity for O2

Explain the conformation change of Hb induced by O2.

- Upon O2 binding by Hb, the Fe2+ atom moves closer to the plane of the heme (as if the O2 is drawing the heme iron into the plane) - This trivial change causes many biological consequences - As Fe2+ moves, it drags His F8 and the F helix with it - This change is transmitted to the subunit interfaces, where conformation changes lead to rupture of salt bridges

Chapter 15 Essential Questions:

- What are the properties of regulatory enzymes? - How do regulatory enzymes sense the momentary needs of cells? - What molecular mechanisms are used to regulate enzyme activity?

Explain how oxygen binding by Hb induces a quaternary structure change.

- When deoxy-Hb crystals are exposed to O2, the shatter (evidence of a large-scale structural change) - One alpha-beta pair (chains) in Hb moves relative to the other by 15 degrees upon O2 binding - This shift is induced by movement of Fe by 0.039 nm when O2 binds

Symmetry model for allosteric regulation:

- When there is no substrate present, the equilibrium favors the right (T) - The substrate binds to R (R is in small amounts at first-- refer to sigmoidal curve) - When substrate/activator binds to R, the equilibrium will shift left to make more of the R form of enzyme (refer to sigmoidal curve). With more R produced, more substrate/activator is able to bind. - When inhibitor binds to T, the equilibrium shifts to the right.

Differentiate between MM and MWC kinetics.

- When you add a lot of activator to R, you essentially are getting rid of the equilibrium found in MWC model, therefore adding a lot of activator resembles MM kinetics - Addition of A shifts curve to left (MM), addition of I shits curve to right (MWC)

15.2 - What are the general features of allosteric regulation?

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15.3 - Can allosteric regulation be explained by conformational changes in proteins?

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15.4 - What kinds of covalent modification regulate the activity of enzymes?

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15.5 - Is the activity of some enzymes controlled by both allosteric regulation and covalent modification?

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END OF LECTURE 2

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Special focus: hemoglobin and myoglobin.

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Explain how Fe2+ is coordinated by His F8 in heme.

-Iron interacts with six ligands in Hb and Mb - Four of these are the N atoms of the porphyrin - A fifth ligand is donated by the imidazole side chain of His F8 (this residue is on the sixth or F helix, and it is the 8th residue in the helix) - When Mb or Hb bind oxygen, the O2 molecule adds to the heme iron as the sixth ligand

Do you understand...

1. Allosteric regulation? 2. Allosteric models? 3. Glycogen phosphorylase and allosteric regulation/covalent modification? 4. Myoglobin and hemoglobin?

What are the two main ways to regulate enzyme activity?

1. increase or decrease the number of enzyme molecules 2. increase or decrease the intrinsic activity of each enzyme molecule

Why does AMP activate glycogen phosphorylase?

A lot of AMP signals that there is not a lot of ATP present. Therefore, the energy stored in glycogen is needing to be broken down by glycogen phosphorylase to yield ATP through glycolysis.

What is the active form of an enzyme called?

The active form of the enzyme is designated that R state.

What factors influence the rate of an enzymatic reaction?

The availability of substrates and cofactors usually determines how fast the enzyme reaction goes.

What is a zymogen?

Zymogens are inactive precursors of enzymes. Typically proteolytic cleavage produces the active enzyme. Proinsulin is an example of a zymogen.

What would happen without 2,3-BPG?

Without 2,3-BPG, Hb would be saturated with oxygen and would not release within tissues.

Explain how sickle cell anemia is caused.

- Sickle cells are abnormally shaped red blood cells (crescent shaped) - The sickle cells pass less freely through the capillaries, impairing circulation and causing tissue damage - A single amino acid substitution in the beta-chains of Hb causes sickle-cell anemia

15.1 - What factors influence enzymatic activity?

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What is the allosteric activator of glycogen phosphorylase?

AMP is an allosteric activator of glycogen phosphorylase.

In the glycogen phosphorylase reaction, what promotes the conversion of T to R?

AMP promotes the conversion of T to R.

What are the allosteric inhibitors of glycogen phosphorylase?

ATP and glucose-6-P are allosteric inhibitors of glycogen phosphorylase.

In the glycogen phosphorylase reaction, what promotes the conversion of R to T?

ATP, glucose-6-P, and caffeine favor the conversion of R to T.

Polymerization of Hb S figure:

Adding a hydrophobic "bump" to Hb S causes "clumping" of Hb S units, which leads to the negative effects of sickle cell anemia.

What happens to H+ binding when you increase O2 binding?

Binding of O2 diminishes H+ binding.

What happens to O2 binding when you increase H+ binding?

Binding of protons diminishes O2 binding.

How are changes in enzyme amounts regulated?

Changes in enzyme amounts are typically regulated via gene expression and protein degradation.

How are changes in the intrinsic activity of enzyme molecules achieved?

Changes in the intrinsic activity of enzyme molecules are achieved principally by allosteric regulation or covalent modification.

What is chymotrypsinogen?

Chymotrypsinogen is an inactive zymogen.

What is cooperativity, and how is it achieved (MWC model)?

Cooperativity is achieved because S binding increases the population of R, which increases the sites available to S.

Covalent modification converts the enzyme from what form to what form?

Covalent modification converts the enzyme from an allosterically regulated form (b) into an active form (a). Covalent modification overrides the allosteric regulation.

Explain what the "tower helix" is in the structure of glycogen phosphorylase.

Each of the two subunits in glycogen phosphorylase contributes a "tower helix" (residues 262 - 278) to the subunit-subunit interface - In the dimer, the tower helices extend from their respective subunits and pack against each other

Hb F versus Hb A kinetics curves:

Fetal Hb has a higher affinity for O2 because 2,3-BPG binds less tightly to Hb due to the Ser in place of His at position 143.

What two factors determine the amount of enzyme present at any moment?

Genetic regulation and enzyme synthesis and decay determines the amount of enzyme present at any moment.

Why does glucose-6-P inhibit glycogen phosphorylase?

Glucose-1-P (product of glycogen phosphorylase) can be converted to glucose-6-P; the second intermediate of glucose-6-P can be used in glycolysis to yield energy. So, if we already have glucose-6-P, we do not need to produce glucose-1-P by breakdown of glycogen via glycogen phosphorylase, and therefore the presence if glucose-6-P acts as an inhibitor.

What happens when cellular energy reserves are low (high concentrations of AMP and low concentrations of ATP and glucose-6-P)?

Glycogen catabolism is stimulated.

What model (MWC or KNF) does glycogen phosphorylase conform to?

Glycogen phosphorylase conforms to the MWC model.

What is hemoglobin?

Hemoglobin is a tetrameric oxygen transport protein. Allosteric regulation can occur in hemoglobin (multiple subunits).

How do subunits of allosteric enzymes communicate?

Interaction of one subunit of an allosteric enzyme with its substrate (or its effectors) is communicated to the other subunits of the enzyme through intersubunit interactions.

Myoglobin and hemoglobin structures:

Myoglobin (one subunit); hemoglobin (four subunits). Red is heme; 1 heme in myoglobin, and 4 hemes in hemoglobin.

What are the oxygen binding curves for hemoglobin and myoglobin?

Myoglobin binds tighter to oxygen (hyperbolic, MM curve). Hemoglobin travels to lungs to pick up oxygen. It transports the O2 to myoglobin to be stored for usage by the muscles. As it travels, the level of saturation becomes lesser and lesser due to the binding of oxygen by myoglobin from hemoglobin. Hemoglobin = allosteric regulation (sigmoidal curve). It is cooperative. The binding of O2 to the first subunit makes binding of O2 to the other subunits more favorable.

What is myoglobin?

Myoglobin is a monomeric oxygen-storage protein. Myoglobin does not go under allosteric regulation (only one subunit).

What is special about 2,3-BPG?

Negative charges interact with 8 positive charges in the cavity; 2 Lys, 4 His, 2 N-termini

What is phosphoprotein phosphatase?

Phosphoprotein phosphatases catalyze the reverse reaction -- removing phosphoryl groups from proteins.

What is the most prominent form of covalent modification in cellular regulation?

Reversible phosphorylation, which is able to regulate enzyme activity.

What is the inactive form of an enzyme called?

The inactive form of the enzyme is denoted the T state.

Which form (phosphorylase a or b) is much less sensitive to allosteric regulation?

The phosphorylated form (a form) is much less sensitive to allosteric regulation than the b form.

Why does ATP inhibit glycogen phosphorylase?

The purpose of glycogen phosphorylase is to cleave a glucose unit off of glycogen so that it can be used in glycolysis in order to make energy. So, if we already have ATP, we don't need more of it, and so the presence of ATP should inhibit the glycogen phosphorylase reaction.

What happens when ATP and glucose-6-P are abundant in the presence of glycogen phosphorylase?

When ATP and glucose-6-P are abundant, glycogen breakdown is inhibited.

Figure on how H+ promotes dissociation of O2 binding by Hb:

When you workout, your body produces lactic acid (H+ ions build up, causing lower pH levels). These lower pH levels promote dissociation of O2 binding by Hb, which means O2 is binding to Mb to be used by the muscles, which is good.

How do ligand-induced conformational changes occur? (KNF sequential model)

a) S binding can, by induced fit, cause a conformational change in the subunit to which it binds b) If subunit interactions are tightly coupled, binding of S to one subunit may cause the the other subunit to assume a conformation having a greater or lesser affinity for S. That is, the ligand-induced conformational change in one subunit can affect the adjoining subunit(s).

Figure on how glycogen phosphorylase activity is regulated allosterically:

a) The response to the concentration of the substrate (Pi) b) ATP and glucose-6-P are feedback inhibitors c) AMP is a positive effector. It binds at the same site as ATP. More like MM kinetics.

Fetal hemoglobin has ___ affinity for oxygen than does adult hemoglobin because it has ___ affinity for 2,3-BPG.

higher; lower

Oxygen binding to myoglobin is ___ by pH whereas decreasing pH ____ the affinity of hemoglobin for oxygen. This is known as ___ effect.

unaffected; decreases; Bohr