Enzyme Regulation Biochemistry by Reginald Garrett and Charles Grisham Chapter 15 Enzyme Regulation Biochemistry by Reginald Garrett and Charles Grisham

Essential Question 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?

Outline of Chapter 15 What Factors Influence Enzymatic Activity? What Are the General Features of Allosteric Regulation? Can a Simple Equilibrium Model Explain Allosteric Kinetics? Is the Activity of Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification?

15.1 – What Factors Influence Enzymatic Activity? The activity displayed by enzymes is affected by a variety of factors, some of which are essential to the harmony of metabolism Two of the more obvious ways to regulate the amount of activity are To increase or decrease the number of enzyme molecule (enzyme level) To increase or decrease the activity of each enzyme molecule (enzyme activity)

A general overview of factors influencing enzyme activity includes the following considerations Rate depends on substrate availability Rate slows as product accumulates Genetic controls (transcription regulation) - induction and repression (enzyme level) Allosteric effectors may be important Enzymes can be modified covalently Zymogens, isozymes and modulator proteins may play a role

Figure 15.1 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes (protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The -OH on the interconvertible enzyme represents an -OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group.

Phosphorylation Adenylylation ADP-ribosylation

A general overview of factors influencing enzyme activity includes the following considerations Rate depends on substrate availability Rate slows as product accumulates Genetic controls (transcription regulation) - induction and repression (enzyme level) Allosteric effectors may be important Enzymes can be modified covalently Zymogens, isozymes and modulator proteins may play a role

Zymogens Figure 15.2 Proinsulin is an 86-residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges.

Figure 15.3 The proteolytic activation of chymotrypsinogen.

Figure 15. 4 The cascade of activation steps leading to blood clotting Figure 15.4 The cascade of activation steps leading to blood clotting. The intrinsic and extrinsic pathways converge at Factor X, and the final common pathway involves the activation of thrombin and its conversion of fibrinogen into fibrin, which aggregates into ordered filamentous arrays that become cross-linked to form the clot. Serine protease: Kallikrein VIIa IXa Xa XIa XIIa Thronbin

formation of a blood clot. Rich in negative charge formation of a blood clot.

Isozymes Figure 18.30 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction.

Figure 15. 5 The isozymes of lactate dehydrogenase (LDH) Figure 15.5 The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobic and produces pyruvate from glucose via glycolysis (Chapter 18). It needs LDH to regenerate NAD+ from NADH so glycolysis can continue. The lactate produced is released into the blood. The muscle LDH isozyme (A4) works best in the NAD+-regenerating direction. Heart tissue is aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate to fuel the citric acid cycle to obtain energy. The heart LDH isozyme (B4) is inhibited by excess pyruvate so the fuel won’t be wasted.

Modulator proteins are another way that cells mediate metabolic activity cAMP-dependent protein kinase Phosphoprotein phosphatase inhibitor-I

Figure 15.6 Cyclic AMP- dependent protein kinase (also known as PKA) is a 150- to 170-kD R2C2 tetramer in mammalian cells. The two R (regulatory) subunits bind cAMP (KD = 3 x 10-8 M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits are enzymatically active as monomers. Cyclic AMP-dependent protein kinase is shown complexed with a pseudosubstrate peptide (red).  This complex also includes ATP (yellow) and two Mn2+ ions (violet) bound at the active site.

Figure 15.1 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes (protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The -OH on the interconvertible enzyme represents an -OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group.

15.2 – What Are the General Features of Allosteric Regulation? Action at "another site" Allosteric regulation acts to modulate enzymes situated at key steps in metabolic pathways A  B  C  D  E  F E, the essential end product, inhibits enzyme 1, the first step in the pathway This phenomenon is called feedback inhibition or feedback regulation Enz 1 Enz 2 Enz 3 Enz 4 Enz 5

Regulatory enzymes have certain exceptional properties Their kinetics do not obey the Michaelis-Menten equation Their v versus [S] plots yield sigmoid- or S-shaped curve A second-order (or higher) relationship between v and [S] Substrate binding is cooperative

Figure 15. 7 Sigmoid v versus [S] plot Figure 15.7  Sigmoid v versus [S] plot. The dotted line represents the hyperbolic plot characteristic of normal Michaelis - Menten-type enzyme kinetics.

Regulatory enzymes have certain exceptional properties Their kinetics do not obey the Michaelis-Menten equation Inhibition of a regulatory enzyme by a feedback inhibitor does not conform to any normal inhibition pattern- Allosteric inhibition Some effector molecules exert negative effects on enzyme activity, other effectors show stimulatory, or positive, influences on activity Oligomeric organization The regulatory effects exerted on the enzyme’s activity are achieved by comformational changes occurring in the proetin when effector metabolites bind

15.3 – Can a Simple Equilibrium Model Explain Allosteric Kinetics? 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 (R or T) T state predominates in the absence of substrate S R0  T0 L= T0 / R0 L is assume to be large (T  R) S binds much tighter to R than to T

Figure 15.8  Monod - Wyman - Changeux (MWC) model for allosteric transitions. Consider a dimeric protein that can exist in either of two conformational states, R or T. Each subunit in the dimer has a binding site for substrate S and an allosteric effector site, F. The promoters are symmetrically related to one another in the protein, and symmetry is conserved regardless of the conformational state of the protein. The different states of the protein, with or without bound ligand, are linked to one another through the various equilibria. Thus, the relative population of protein molecules in the R or T state is a function of these equilibria and the concentration of the various ligands, substrate (S), and effectors (which bind at FR or FT). As [S] is increased, the T/R equilibrium shifts in favor of an increased proportion of R-conformers in the total population (that is, more protein molecules in the R conformational state).

Although the relative [R0] concentration is small, S will bind ‘only’ to R0, forming R1 S-binding drives the conformation transition, T0  R0 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

Figure 15. 9 The Monod - Wyman - Changeux model Figure 15.9  The Monod - Wyman - Changeux model. Graphs of allosteric effects for a tetramer (n = 4) in terms of Y, the saturation function, versus [S]. Y is defined as [ligand-binding sites that are occupied by ligand]/[ total ligand-binding sites]. (a) A plot of Y as a function of [S], at various L values. (b) Y as a function of [S], at different c, where c = KR/KT. (When c = 0, KT is infinite.) (Adapted from Monod, J., Wyman, J., and Changeux, J.-P., 1965. On the nature of allosteric transitions: A plausible model. Journal of Molecular Biology 12:92.)

Molecules that influence the binding of something other than themselves are heterotropic effectors Positive heterotropic effectors or allosteric avtivators negative heterotropic effectors or allosteric inhibitors

Figure 15.10  Heterotropic allosteric effects: A and I binding to R and T, respectively. The linked equilibria lead to changes in the relative amounts of R and T and, therefore, shifts in the substrate saturation curve. This behavior, depicted by the graph, defines an allosteric “K” system. The parameters of such a system are: (1) S and A (or I) have different affinities for R and T and (2) A (or I) modifies the apparent K0.5 for S by shifting the relative R versus T population.

K system and V system are two different forms of the MWC model In K system: The concentration of S giving half-maximal velocity, defined as K0.5, changes in response to effectors Vmax is constant In V system K0.5 is constant Vmax change V versus [S] plots are hyperbolic rather than S-shaped

15.4 Is the Activity of Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification? Allosteric Regulation and Covalent Modification Glycogen phosphorylase cleaves glucose units from nonreducing ends of glycogen A phosphorolysis reaction Muscle glycogen phosphorylase is a dimer of identical subunits, each with PLP covalently linked There is an allosteric effector site at the subunit interface

Figure 15.12 The glycogen phosphorylase reaction. Figure 15.13 The phosphoglucomutase reaction.

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues) Each subunit contains a pyridoxal phosphate cofactor covalently linked (Lys-680) An active site An allosteric effector site near the subunit interface A regulatory phosphorylation site (Ser-14) A glycogen binding site A tower helix (residues 262 to 278)

Figure 15.14  (a) The structure of a glycogen phosphorylase monomer, showing the locations of the catalytic site, the PLP cofactor site, the allosteric effector site, the glycogen storage site, the tower helix (residues 262 through 278), and the subunit interface. (b) Glycogen phosphorylase dimer.

Allosteric Regulation of GP Cooperativity in substrate binding (15.15a) Inorganic phosphate (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)

Figure 15. 15 v versus S curves for glycogen phosphorylase Figure 15.15 v versus S curves for glycogen phosphorylase. (a) The sigmoid response of glycogen phosphorylase to the concentration of the substrate phosphate (Pi) shows strong positive cooperativity. (b) ATP is a feedback inhibitor that affects the affinity of glycogen phosphorylase for its substrates but does not affect Vmax. (Glucose-6-P shows similar effects on glycogen phosphorylase.) (c) AMP is a positive heterotropic effector for glycogen phosphorylase. It binds at the same site as ATP. AMP and ATP are competitive. Like ATP, AMP affects the affinity of glycogen phosphorylase for its substrates, but does not affect Vmax.

Figure 15.16  The mechanism of covalent modification and allosteric regulation of glycogen phosphorylase. The T states are blue and the R states blue-green.

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 15.18)

Figure 15.17 In this diagram of the glycogen phosphorylase dimer, the phosphorylation site (Ser14) and the allosteric (AMP) site face the viewer. Access to the catalytic site is from the opposite side of the protein. The diagram shows the major conformational change that occurs in the N-terminal residues upon phosphorylation of Ser14. The solid black line shows the conformation of residues 10 to 23 in the b, or unphosphorylated, form of glycogen phosphorylase. The conformational change in the location of residues 10 to 23 upon phosphorylation of Ser14 to give the a (phosphorylated) form of glycogen phosphorylase is shown in yellow. Note that these residues move from intrasubunit contacts into intersubunit contacts at the subunit interface. [Sites on the two respective subunits are denoted, with those of the upper subunit designated by primes (‘).] (Adapted from Johnson, L. N., and Barford, D., 1993. The effects of phosphorylation on the structure and function of proteins. Annual Review of Biophysics and Biomolecular Structure 22:199-232.)

Figure 15.18  The hormone-activated enzymatic cascade that leads to activation of glycogen phosphorylase.

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

Figure 15.19  The adenylyl cyclase reaction yields 3',5' -cyclic AMP and pyrophosphate. The reaction is driven forward by subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase.

Figure 15.20 Hormone (H) binding to its receptor (R) creates a hormone;receptor complex (H:R) that catalyzes GDP-GTP exchange on the a -subunit of the heterotrimer G protein (Gabg ), replacing GDP with GTP. The Ga -subunit with GTP bound dissociates from the bg -subunits and binds to adenylyl cyclase (AC). AC becomes active upon association with Ga :GTP and catalyzes the formation of cAMP from ATP. With time, the intrinsic GTPase activity of the Ga -subunit hydrolyzes the bound GTP, forming GDP; this leads to dissociation of Ga :GDP from AC, reassociation of Ga with the bg subunits, and cessation of AC activity. AC and the hormone receptor H are integral plasma membrane proteins; Ga and Gbg are membrane-anchored proteins.

A classic example of allostery Hemoglobin A 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 as of 141 residues, 2 bs of 146

Figure 15.21 O2-binding curves for hemoglobin and myoglobin.  

Hemoglobin Function Hb must bind oxygen in lungs and release it in capillaries Adjacent subunits' affinity for oxygen increases This is called positive cooperativity

Competition between oxygen and H+ 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 15.33

Figure 15.33  The oxygen saturation curves for myoglobin and for hemoglobin at five different pH values: 7.6, 7.4, 7.2, 7.0, and 6.8.

Carbon dioxide diminishes oxygen binding 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

Figure 15.34 Oxygen-binding curves of blood and of hemoglobin in the absence and presence of CO2 and BPG. From left to right: stripped Hb, Hb + CO2, Hb + BPG, Hb + BPG + CO2, and whole blood.