Lecture 12

Lecture 12 Enzyme regulation

What Factors Influence Enzymatic Activity?

• Two of the more obvious ways to regulate the amount of activity are

1. To increase or decrease the number of enzyme molecule (enzyme level)

2. To increase or decrease the activity of each enzyme molecule (enzyme activity)

• A general overview of factors influencing enzyme activity includes the following considerations

1. Rate depends on substrate availability 2. Rate slows as product accumulates 3. Allosteric effectors may be important 4. Enzymes can be modified covalently 5. Genetic controls (transcription regulation) -

induction and repression (enzyme level)6. Zymogens, isozymes and modulator proteins

may play a role




may play a role

Non-covalent InteractionsSubstrate availability

• Non-regulatory enzymes generally exhibit hyperbolic kinetics (Michaelis-Menton)

• At low substrate concentration, reaction rate proportional to substrate concentration

• Regulatory enzymes generally exhibit sigmoidal kinetics (positive cooperativity)

• Changes of substrate concentrations at normal physiological levels greatly alter reaction rate

• Regulatory enzymes are usually the enzymes that are the rate-limiting step, in a pathway, meaning that after this step a particular reaction pathway will go to completion




may play a role

Non-covalent InteractionsAllosteric Regulation

• Binding of allosteric effectors at allosteric sites affect catalytic efficiency of the enzyme


• Allosteric Activators– Decrease Km (increases the enzyme bindin

g affinity)– Increases Vmax (increases the enzyme cata

lytic efficiency)


• Allosteric Inhibitors– Increases Km (decreases enzyme binding affi

nity)– Decreases Vmax (decreases enzyme catalytic

efficiency)

Molecules that act as allosteric effectors

• End products of pathways– Feedback inhibition

• Substrates of pathways– Feed-forward activators

• Indicators of Energy Status– ATP/ADP/AMP– NAD/NADH– Citrate & acetyl CoA

Allosteric Example• Feedback Inhibition - This occurs

when an end-product of a pathway accumulates as the metabolic demand for it declines.

• This end-product in turn binds to the regulatory enzyme at the start of the pathway and decreases its activity - the greater the end-product levels the greater the inhibition of enzyme activity.

Metabolic Pathway Product/ Feedback Inhibition

Feed-forward activators Phosphofructokinase ( PFK)

Fructose-6-P + ATP -----> Fructose-1,6-bisphosphateFructose-1,6-bisphosphate + ADPADP

•PFK catalyzes 1st committed step in glycolysis (10 steps total)

(Glucose + 2ADP + 2 NAD+ + 2Pi 2pyruvate + 2ATP + 2NADH)

•ADP is an allosteric activator of PFK

Allosteric modulators bind to site other than the active site

Fructose-6-P + ATP -----> Fructose-1,6-bisphosphateFructose-1,6-bisphosphate + ADPADP

ADPADP

Allosteric Activator (ADP) binds distal to active site

PFK exists as a homotetramer in bacteria and mammals

Vo vs [S] plots give sigmoidal curve for at least one substrate

Binding of this allosteric inhibitor or this activator does not effect Vmax, but does alter Km

Allosteric enzyme do not follow M-M kinetics

Activator can shift hyperbolic (as if there were no T state)

Sample questions• Two curves showing the rate versus substrate concentration are sho

wn below for an enzyme‐catalyzed reaction. One curve is for the reaction in the presence of substance X. The other curve is for data in the absence of substance X. Examine the curves and tell which statement below is true.

• A) The catalysis shows Michaelis‐Menten kinetics with or without X.• B) X increases the activation energy for the catalytic reaction.• C) X could be a competitive inhibitor.• D) X is an activator of the enzyme.

Sample questionsAllosteric enzymes are• A.similar to simple enzyme• B.smaller than simple enzyme• C.larger and more complex than simple enzyme• D.smaller than simple enzyme but not complex

Which statement is false about allosteric regulation?• A. It is usually the mode of regulation for the last step in reaction pathways s

ince this step produces the final product.• B. Cellular response is faster with allosteric control than by controlling enzy

me concentration in the cell.• C. The regulation usually is important to the conservation of energy and mat

erials in cells.• D. Allosteric modulators bind non-covalently at sites other than the active sit

e and induce conformational changes in the enzyme.

Sample questions

Allosteric modulators seldom resemble the substrate or product of the enzyme. What does this observation show?

• A) Modulators likely bind at a site other than the active site.

• B) Modulators always act as activators.• C) Modulators bind non-covalently to the enzyme.• D) The enzyme catalyzes more than one reaction.

Sample questions

• Some enzymatic regulation is allosteric. In such cases, which of the following would usuallybe found?

• A) cooperativity• B) feedback inhibition• C) both activating and inhibitoryactivity• D) an enzyme with more than one subunit• E) the need for cofactors

Sample questions

• Describe allosteric regulation of enzyme activity?

An allosteric enzyme is one in which the activity of the enzyme can be controlled by the binding of a molecule to the “allosteric site”, somewhere other than the active site. Thus allosteric control of an enzyme can be classed in two ways. A positive allosteric regulation is the binding of a molecule to the enzyme which increase the rate of reaction. The opposite is a negative allosteric regulation. An example for this is phosphofructokinase, which is promoted by a high AMP concentration, and inhibited by a high ATP concentration.

Non-covalent InteractionsProtein-Protein Interactions

• Calmodulin (CALcium MODULted proteIN)

– Binding of Ca++ to calmodulin changes its shape and allows binding and activation of certain enzymes

Binding of calcium to Calmodulin changes the shape of the protein

Unbound Calmodulin on left

Calcium bound Calmodulin on right. Stars indicate exposed non-polar ‘grooves’ that non-covalently binds proteins

Calmodulin

• Extracellular [Ca] = 5 mM

• Intracellular [Ca] = 10-4 mM– Bound Ca can be released by hormonal actio

n, nerve innervation, light, ….– Released Ca binds to Calmodulin which activ

ates a large number of proteins

Calmodulin plays a role in:

• Muscle contraction• Inflammation• Apoptosis• Memory• Immune response….• Metabolism

– Activates phosphorylase kinase• Stimulates glycogen degradation during exercise




may play a role

Covalent Regulation of Enzyme ActivityPhosphorylation and Dephosphorylation

• Addition or deletion of phosphate groups to particular serine, threonine, or tyrosine residues alter the enzymes activity

Enzymes regulated by covalent modification are called interconvertible enzymes.

The enzymes (protein kinase and protein phosphatase) 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.

Covalent Regulation of Enzyme ActivityEnzyme Cascades

• Enzymes activating enzymes allows for amplification of a small regulatory signal

Sample questions

• Which statement is false about covalent modification?• A) It is reversible.• B) It is slower than allosteric regulation.• C) It is irreversible.• D) Phosphorylation is a common covalent modification.

Sample questions

Protein kinases are enzymes that act on other enzymes by adding phosphates groups. When the enzyme is phosphorylated, it changes its activity (it becomes more or less active, depending on the enzyme). This regulatory mechanism of enzymatic activity is called:

• A) Allosteric Control• B) Competitive inhibition• C) Covalent Modification• D) Isozymes Modification • E) Zymogen activation




may play a role

Changes in Enzyme Abundance

• Inducible vs Constitutive Enzymes

• Induction is caused by increases in rate of gene transcription.– Hormones activate transcriptional factors

• Increase synthesis of specific mRNA• Increase synthesis of specific enzymes

• Induction (an increase caused by an effecter molecule) of enzyme synthesis is a common mechanism - this can manifest itself at the level of gene expression, RNA translation, and post-translational modifications. The actions of many hormones and/or growth factors on cells will ultimately lead to an increase in the expression and translation of "new" enzymes not present prior to the signal.

Regulation of Enzyme Concentrations: Induction

Regulation of Enzyme Concentrations: Degradation

• The degradation of proteins is constantly occurring in the cell.

• Proteolytic degradation is an irreversible mechanism.

• Protein degradation by proteases is compartmentalized in the cell in the lysosome (which is generally non-specific), or in macromolecular complexes termed proteasomes.

• Degradation by proteasomes is regulated by a complex pathway involving transfer of a 76 aa polypeptide, ubiquitin, to targeted proteins. Ubiquination of protein targets it for degradation by the proteasome. This pathway is highly conserved in eukaryotes, but still poorly understood

Regulation of Enzyme Concentrations: Degradation (cont)




may play a role

Covalent Regulation of Enzyme ActivityLimited Proteolysis

• Specific proteolysis can activate certain enzymes and proteins (zymogens)– Digestive enzymes– Blood clotting proteins– Peptide hormones (insulin)

Regulation signaling ：

Hormones, Receptors, and Communication Between Cells

• Hormones– chemical signals that coordinate metabolism

• Hormone Receptors– Target tissues– Specific binding– Types

• Intracellular receptors• Cell-surface receptors


• Intracellular receptors

• lipid soluble hormones• Steroid hormones, vitamin D, retinoids, thyroxine

• Bind to intracellular protein receptors – This binds to regulatory elements by a gene– Alters the rate of gene transcription

• Induces or represses gene transcription


Intracellular Receptors


• Cell-surface receptors– Water soluble hormones

• Peptide hormones (insulin), catecholamines, neurotransmitters

• Three class of cell-surface receptors– Ligand-Gated Receptors– Catalytic Receptors– G Protein-linked Receptors

Cell Surface Receptors

Ligand-Gated Receptors

• Binding of a ligand (often a neurotransmitter) affects flow of ions in/out of cell

• Gamma-amino butyric acid (GABA) binds and opens chloride channels in the brain– Valium (anti-anxiety drug) reduces the amount of GABA

required to open the chloride channels

Cell-Surface Receptors

Catalytic Receptors

• Binding of hormone activates tyrosine kinase on receptor which phosphorylates certain cellular proteins

• Insulin receptor is a catalytic receptor with TYR Kinase activity

Cell-Surface Receptors

G Protein-Linked Receptors

• Binding of hormone activates an enzyme via a G-protein communication link.

• The enzymes produces intracellular messengers– Signal transduction– Second messengers activate p

rotein kinases

Intracellular Messengers:

Signal Transduction Pathways

• Cyclic AMP (cAMP)

• Diacylglycerol (DAG) & Inositol Triphosphate (IP3)

• Cyclic GMP (cGMP)

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

G-Protein-Linked Receptors:The cAMP Signal Transduction Pathway

• Two types of G-Proteins• Stimulating G protein (Gs)

– Activate adenylate cyclase

• Inhibitory G proteins (Gi)

– Inhibit adenylate cyclase

G Proteins

• G proteins are trimers – Three protein units

• Alpha• Beta• gamma

• Alpha proteins are different in Gs and Gi

– Both have GTPase activity– Alpha proteins modify adenylate cyclase activity

• AC stimulated by Alpha(s) when activated by a hormone• AC Inhibited by Alpha(I) when activated by other hormones

Family of G Proteins

• Binding of hormones to receptors causes: – GTP to displace GDP – Dissociation of alpha pro

tein from beta and gamma subunits

– activation of the alpha protein

– Inhibition or activation of adenylate cyclase

– GTPase gradually degrades GTP and inactivates the alpha protein effect (clock)

cAMP

The cAMP Signal Transduction Pathway

• cAMP – intracellular messenger– Elevated cAMP can either activate or inhibit regulator

y enzymes• cAMP activates glycogen degradation• cAMP inhibits glycogen synthesis

• [cAMP] affected by rates of synthesis and degradation– Synthesis by adenylate cyclase– Degradation by phosphodiesterase

• Stimulated by insulin• Inhibited by caffeine

What does cAMP do?Activation of Protein Kinase A by cAMP

• Protein kinase A– Activates or inhibits several enzymes – Inactive form: regulatory+catalytic subunits as

sociated– Active form: binding of cAMP disassociates su

bunits

Clinical Case ： Cholera

• Severe and rapid diarrheal disease– Caused by Vibrio cholerae– Commonly shock after 4-12 hrs aft

er first symptoms, death 18 hrs – several days without rehydration therapy (subject can lose up to 20 liters of fluids)

– Source is commonly contaminated water

Choleramechanism of action

• V. cholarae produces protein that attaches to intestinal epithelial cells– Delivery subunit B (blue) facilitates entr

y of subunit A into cell

• Subunit A catalyzes ADP-ribosylation of the alpha-s subunit of Gs-protein

Clinical Case

• V. cholerae toxin affects alpha-S subunit– Inactivates GTPase– Alpha-S subunit permanently active

• Stimulates adenylate cyclase– Overproduces cAMP– stimulates protein kinase – Phosphorylation of membrane ion transpo

rt proteins – massive losses of Na, Cl, K, HCO3

Hypothetical link to cystic fibrosis

• Cystic fibrosis characterized by– Salty sweat– Very thick mucous

• Homozygous genetic defect to chloride transport to mucous– Decreased chloride results in less water following due

to osmosis, leading to thicker mucous

• Heterozygous mutation (normal mucous) has transport protein resistant to effects of cholera toxin ?






DAG & IP3

Phosphotidylinositol Signal Transduction Pathway• Hormone activation of phospholipase C

– Via Gp protein• Phospholipase C hydrolyzes membrane phospholipids (p

hosphotidyl inositol) to produce DAG and IP3

• IP3 stimulates release of Ca from ER• Protein kinase C activated by DAG and calcium






cGMPThe cGMP Signal Transduction Pathway

• cGMP effects:

• lowering of blood pressure & decreasing CHD risk– Relaxation of cardiac muscle– Vasodilation of vascular smooth muscle– Increased excretion of sodium and water by kidney– Decreased aggregation by platelet cells


• Two forms of guanylate cyclase• Membrane-bound

• Activated by ANF (atrial natriuretic factor)– ANF released when BP elevated

• Cytosolic• Activated by nitric oxide• NO produced from arginine by NO synthase (activated by C

a)– Nitroglycerine slowly produces NO, relaxes cardiac and vascula

r smooth muscle, reduces angina

• cGMP activates Protein Kinase G– Phosphorylates smooth muscle proteins


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Lecture 12