Enzymes - Detailed Study Notes

Enzymes - Chapter 6

Objectives

• Describe the shared characteristics of enzymes and how they differ from chemical catalysts.
• Investigate the relationship between free energy and reaction properties, including the influence of enzymes on these parameters.
• Discriminate binding and chemical effects on enzyme catalysis.
• Apply the plots and equations used to quantify enzyme kinetics.
• Explain the catalytic mechanism of serine protease.
• Elucidate the molecular basis of the different classes of reversible enzyme inhibition.
• Examine the mechanisms and logic of biological regulation of enzymes.
• Text Readings: Stryer 2nd or 3rd Edition All of Chapters 6, 7 and 8

Enzymes - Introduction

• Life depends on the ability to efficiently and selectively catalyze chemical reactions.
• Most biomolecules are very stable with rates of uncatalyzed transformations that are too slow to permit life.
• Enzymes provide a mechanism for acceleration, regulation, and coordination of these reactions.
• The most striking feature about enzymes is their catalytic power and specificity.
• Side reactions leading to useless or dangerous molecules must be avoided.
• Some enzymes are information sensors as well as catalysts.

Enzymes - Vitalism

• Eduard Buchner demonstrated that dead yeast still convert sugars into alcohol, indicating the reactions of life were separate from life.
• There was a factor in yeast catalyzing the reaction; the term "enzyme" is from the Greek "in yeast".
• This work won Eduard Buchner the Nobel Prize, ten years before he was killed in WWI.
• Originally biochemical reactions were believed to be inseparable from life.
• Vitalism is the belief that living things are fundamentally different from non-living things; that they contain some non-physical element and are governed by different principles that inanimate objects.
• Vitalism had some famous supporters, including Louis Pasteur.

Enzymes - Co-Enzymes and Co-Factors

• Proteins are well suited to form a variety of complex three-dimensional structures that enable binding of a variety of substrates.
• For some enzymes, the protein component alone is fully active.
• Other enzymes require co-factors (inorganic ions ($Mg^{2+}$, $Fe^{2+}$, etc.)) or co-enzymes (complex organic molecules (vitamins)) for activity.
• A co-enzyme or co-factor that is tightly associated with the enzyme is called a prosthetic group (the difference is the degree of association).
• Different enzymes that use the same coenzyme usually perform similar types of reactions.
• $Apoenzyme + Co-factor/Co-enzyme = Holoenzyme$

Catalysts - General

Catalysts:

• lower the amount of energy required for a reaction to proceed.
• sped up attainment of equilibrium but do not change equilibrium.
• are unchanged by the reaction; recycled to participate in another reaction.
• Enzymes offer incredible catalytic power in the rate enhancements they provide.

Catalysts - Enzymes vs Chemical Catalysts

1. Speed: Enzymes are often much faster than chemical catalysts, some approaching catalytic perfection.
2. Conditions: Many chemical catalysts that require extremes of temperature, pressure and pH while enzymes function under physiological conditions.
3. Specificity: Enzymes have a higher degree of specificity (including stereospecificity) than most chemical catalysts. This includes specificity for what they act upon and what they produce.
4. Regulation: Unlike chemical catalysts, many enzymes are responsiveness to the dynamic needs of the cell and organism.

Enzymes - Circe Effect

• Enzyme rates of catalysis can approach the physical limit of rates of diffusion of molecules in solution.
• Some enzymes have rate-determining steps that are roughly as fast as the binding of substrates to the enzymes.
• Some enzymes are able to catalyze reaction faster than predicted by diffusion-control limits.
• This is called the Circe effect, named after a figure in Greek mythology who was renowned for her ability to draw her enemies to her, then transform them into animals.

Enzymes - Equilibrium and ES Complex

• Enzymes catalyze the interconversion of substrate and product.
  $E + S \leftrightharpoons ES \leftrightharpoons E + P$
• Substrate (S): the molecule acted upon by the enzyme.
• Product (P): the molecule produced by the enzyme.
• Active Site: the portion of enzyme (E) responsible for binding the substrate to formation of an enzyme-substrate (ES) complex.

Enzymes - The Active Site

1. The active site is a 3D cleft formed from different parts of the polypeptide chain.
2. The active site represents just a small part of the enzyme.
3. Active sites are unique microenvironments.
4. Substrates are bound to enzymes by multiple weak interactions.
5. The specificity of substrate binding depends on the precisely defined arrangement of atoms in the active site.
6. Enzymes and their active sites can be quite flexible.
7. Substrate binding can caused “induced fit” or “conformation selection”.

Enzyme Specificity - Lock-and-key vs Hand-in-Glove

• Lock and Key model: The substrate fits perfectly into the active site of the enzyme, like a key in a lock.
• Hand in Glove (Induced Fit) model: The enzyme's active site changes shape slightly to accommodate the substrate.

Enzymes - Free Energy (Rates and Equilibrium)

1. A reaction is spontaneous only if $\Delta G$ is negative. Spontaneous means the reaction will proceed without the input of energy and the reaction releases energy (exergonic).
2. A reaction cannot take place spontaneously if $\Delta G$ is positive. An input of free energy is required to drive such reactions (endergonic).
3. In a system at equilibrium, there is no net change in the concentrations of the products and reactants, and the $\Delta G$ is zero.
4. The $\Delta G$ of a reaction depends only on the free energy of the product minus the free energy of the reactants. The $\Delta G$ of a reaction is independent of the steps of the transformation.
5. The $\Delta G$ provides no information about the rate of a reaction. A negative $\Delta G$ indicates that a reaction can take place spontaneously but does not signify whether it will proceed at a perceptible rate.

Enzymes - Free Energy (Rates and Equilibrium)

• Activation energy, $\Delta G^{\ddagger}$, between S and P determines the rate at which equilibrium is reached.
• Enzymes provide an alternate, lower-energy pathway between the substrate and product, lowering $\Delta G^{\ddagger}$.
• The relationship between the rate of a reaction and the activation energy is inverse and exponential.
• Difference in free energy between S and P determines the equilibrium of the reaction.
• Enzymes do not influence the difference in free energy between S and P and therefore do not influence the equilibrium.

Enzymes - Rate Enhancements and Equilibrium

• Enzymes provide a lower-energy pathway between the substrate and product, decreasing the activation energy of the transition state and increasing the rate of reaction.
• Enzymes do not affect the difference in free energy between the substrate and product and therefore do not influence the equilibrium of a reaction.

Enzymes - Modes of Enzyme Catalysis

• What are the forces that lower the activation energy?
• Catalytic capabilities of enzymes result from both chemical and binding effects.

Binding Effects

1. Substrate Binding
2. Transition-state Stabilization

Chemical Effects

1. Acid/base catalysis
2. Covalent catalysis

Binding Effects - Reaction Specificity and Catalysis

• Binding of substrate in the active site provides specificity and catalytic power.
• Catalytic mechanisms limited to binding properties can still increase reaction rates by over 10,000-fold.

Binding Effects

1. Substrate Binding
2. Transition-state Stabilization

$E + S \leftrightharpoons ES \leftrightharpoons ETS \leftrightharpoons E + P$

• There is conceptual overlap between substrate binding and transition state stabilization.

Binding Effects - Substrate Binding

Substrate binding promotes reactions by:

1. Reducing entropy (decreased freedom of motion of two molecules in solution).
2. Alignment of reactive functional groups of the enzyme with the substrate.
3. Desolvation of the substrate (removal of water molecules) to expose reactive groups.
4. Distortion of substrates.
5. Induced fit of the enzyme in response to substrate binding.

Binding Effects - Transition-State (TS) Stabilization

• An increased interaction of the enzyme and substrate occurs in the transition-state.
• The essence of catalysis is stabilization of the transition state.
• The enzyme distorts the substrate, forcing it toward the transition state.
• The active site is complementary to the transition-state in shape and chemical character.
• Enzymes may bind their transition states $10^{10}$ to $10^{15}$ times more tightly than their substrates.
• Active site must be similar enough to substrate to ensure specificity, different enough to promote change.

Transition-State Analogs - Competitive Inhibitors

• Transition-state analogs (TSAs) are stable compounds that resemble unstable transition states.
• They have potential therapeutic applications as competitive inhibitors.
• Competitive Inhibitors are molecules that bind to the active site of an enzyme, they tend to resemble the substrate molecule.
• TSAs can bind the active site of a target enzyme active site with high affinity, preventing substrate binding.

Enzymatic Catalysis - Chemical Effects

• After substrate binding, the enzyme can act upon the substrate to promote formation of the product.
• The active site often contains chemically reactive side chains.
• This includes polar, ionizable side chains (triprotics) such as Asp, Glu, His, Cys, Tyr, Lys, Arg, and Ser.
• Two commonly observed mechanisms of chemical catalysis are:
  • Acid/base Catalysis
  • Covalent Catalysis

Chemical Modes of Enzymatic Catalysis - Acid-Base Catalysis

• Reaction acceleration is achieved by catalytic transfer of a proton.
• The side chains of some amino acids can acts as either bases (proton acceptors) or acids (proton donors).
• Histidine, with a pKa near physiological pH, is often involved in acid/base catalysis.
• The pKa of a functional group is influenced by the chemical microenvironment.
• Functional groups of amino acids can have different pKas within the active site which make them more suitable for acid/base catalysis.

Chemical Modes of Enzymatic Catalysis - Covalent Catalysis

• As a part of the reaction mechanism the substrate is covalently bound to the enzyme to form a reactive intermediate.

• In this example, group is transferred from A-X to B in two steps via the covalent complex of X-Enz.

• Covalent catalysis often involves two steps, the first which forms a covalent linkage to the enzyme, the second to regenerate the free enzyme.
  $A-X + E \longrightarrow X-E + A \text{Stage 1}$
  $X-E + B \longrightarrow B-X + E \text{Stage 2}$
  $A-X+ B \longrightarrow B-X + A \text{Overall Reaction}$

Covalent Catalysis - Sucrose Phosphorylase

• Sucrose* + Pi → Fructose + Glucose-1-P (*Sucrose is a disaccharide of glucose and fructose)
• Step one: Glucosyl residue is transferred to enzyme
  $Glucose-Fructose + Enz \longrightarrow Glucosyl-Enz + Fructose$
• Step two: Glucose is transferred to phosphate
  $Glucosyl-Enz + Pi \longrightarrow Glucose 1-phosphate + Enz$

Enzyme Kinetics - General

• Kinetics is the study of the velocity of reactions.
  $Substrate \longrightarrow Product$
• The velocity of a reaction is quantified as the change in concentration of product over time.
  $V = \frac{\Delta[P]}{\Delta t}$
• Enzyme kinetics measured in sunits of concentration over time, for example mmoles/sec or moles/min.

Enzymes Kinetics - Variables that Enzyme Velocity

• As enzymes are proteins, any variable that influences protein structure may influence enzyme activity.
• The activity of enzymes is temperature and pH sensitive.
• Enzymes can have different optimum temperatures and pHs.

Enzymes Kinetics - Variables that Enzyme Velocity

• Enzyme velocities are also influenced by enzyme and substrate concentration.
• For kinetics, we are most interested in the relationship between velocity and substrate concentration.

Kinetics - Initial Velocity (Vo)

• As velocity is defined as the change in product concentration over time, it is necessary to measure product formation before equilibrium is reached.
• Initial velocity ($V_o$) is the velocity at the beginning of an enzyme catalyzed reaction, prior to product accumulation.
• $k<em>1$ and $k</em>{-1}$ represent rapid, non-covalent interactions between enzyme and substrate.
• $k<em>2$ is rate constant of formation of product from ES. $E + S \leftrightharpoons ES \longrightarrow E + P$$V</em>o = [ES]k_2$

Michaelis-Menton Kinetics - Steady State Assumption

• In deriving the Michaelis and Menton equation they worked from the assumption that the rate of formation of the ES complex was equal to the rate of its breakdown.
• This is known as the steady-state assumption.
• Mathematically the steady state assumption states that:
  $[E][S]k<em>1 = [ES]k</em>{-1} + [ES]k_2$
• Rate of formation of the ES complex is $[E][S]k_1$
• Rate of breakdown of the ES complex is $[ES]k<em>{-1} + [ES]k</em>2$
  $E + S \leftrightharpoons ES \longrightarrow E + P$

Michaelis-Menten - Equation and Plot

• The Michaelis-Menten equation and plot describe the relationship between substrate concentration and initial velocity.
  $V<em>o = \frac{V</em>{max}[S]}{K_m + [S]}$
• $K<em>m$ is the concentration of substrate required to reach 1/2 $V</em>{max}$.
• $V_{max}$ is the maximum velocity of the enzyme.

Michaelis-Menten - Km

• $K_m$ is the concentration of substrate required for the enzyme to function at half maximal velocity.
• For many enzymes, $K_m$ provides an accurate approximation of the in vivo substrate concentration.
• This means that most enzymes are usually functioning at about half their maximum velocity.

Michaelis-Menten - Km

• When [S] < K_m, enzymes are highly sensitive to changes in substrate concentration but have very little activity.
• When [S] > K_m, enzymes have high activity but are insensitive to changes in substrate concentration.
• When $[S] = K_m$, enzyme has significant activity and is responsive to changes in substrate concentration.

Michaelis-Menten - Sample Question #1

1. What is the velocity of a reaction when substrate concentration is equal to $K_m$?
   • Step 1: Pick values of $K<em>m$ and $[S]$ that are consistent with the question. Here we can say that $K</em>m$ and $[S]$ are both equal to 2 mM.
   • Step 2: Substitute these values into the equation.
   • Step 3: Express the velocity as a fraction of $V<em>{max}$. $V</em>o = \frac{V<em>{max}[S]}{K</em>m + [S]}$
     $V<em>o = \frac{V</em>{max} \times 2 \text{mM}}{2 \text{mM} + 2 \text{mM}}$
     $V<em>o = \frac{V</em>{max} \times 2 \text{mM}}{4 \text{mM}}$
     $V<em>o = \frac{1}{2}V</em>{max}$

Michaelis-Menten - Sample Question #2

2. What is reaction velocity when substrate concentration is double $K_m$?
   • Step 1: Pick values of $K<em>m$ and $[S]$ that are consistent with the question. Here we can say that $K</em>m$ is equal to 2 mM and $[S]$ is equal to 4 mM.
   • Step 2: Substitute these values into the equation.
   • Step 3: Express the velocity as a fraction of $V<em>{max}$. $V</em>o = \frac{V<em>{max}[S]}{K</em>m + [S]}$
     $V<em>o = \frac{V</em>{max} \times 4 \text{mM}}{2 \text{mM} + 4 \text{mM}}$
     $V<em>o = \frac{V</em>{max} \times 4 \text{mM}}{6 \text{mM}}$
     $V<em>o = \frac{2}{3}V</em>{max}$

Michaelis-Menten - Sample Question #3

3. What is the reaction velocity when substrate concentration is a third of $K_m$?
   • Step 1: Pick values of $K<em>m$ and $[S]$ that are consistent with the question. Here we can say that $K</em>m$ is equal to 3 mM and $[S]$ is equal to 1 mM.
   • Step 2: Substitute these values into the equation.
   • Step 3: Express the velocity as a fraction of $V<em>{max}$. $V</em>o = \frac{V<em>{max}[S]}{K</em>m + [S]}$
     $V<em>o = \frac{V</em>{max} \times 1 \text{mM}}{3 \text{mM} + 1 \text{mM}}$
     $V<em>o = \frac{V</em>{max} \times 1 \text{mM}}{4 \text{mM}}$
     $V<em>o = \frac{1}{4}V</em>{max}$

Kinetics - Lineweaver-Burk Plots

Lineweaver-Burke plots:

• also describe the relationship between $[S]$ and $V_0$.
• are a double-reciprocal plot of $1/V_o$ vs $1/[S]$.
• are a more precise method of analysis of kinetic data.
• are used to determine $V<em>{max}$ and $K</em>m$.
  $\frac{1}{V<em>o} = \frac{K</em>m}{V<em>{max}[S]} + \frac{1}{V</em>{max}}$

Kinetics - Enzyme Turnover Number

Enzyme Turnover Number:

• also called $k_{cat}$
• equals the number of molecules of substrate converted to product per unit time under saturating conditions.
• is calculated by $V<em>{max} / [E</em>t]$.

Reversible Enzyme Inhibition - General

• An inhibitor is a compound that binds to an enzyme to interfere with its activity.
• Inhibitors can prevent formation of ES or the breakdown to E and P.
• Reversible inhibitors bind to the enzyme by non-covalent interactions.
• We will consider two classes of reversible enzyme inhibitors with different mechanisms and kinetic consequences.
  • Competitive
  • Uncompetitive
  • Noncompetitive

Reversible Enzyme Inhibition - Competitive

• Competitive inhibitors resemble the substrate and compete with the substrate for binding the active site.
• The antibiotic sulfanilamide is a competitive inhibitor of a bacterial enzyme that has PABA as a substrate.
• Competitive inhibitors bind only the free enzyme.
• The effect of competitive inhibitors can be overcome with an excess of substrate (washing out).
• $V<em>{max}$ is the same but apparent $K</em>m$ is increased.

Reversible Enzyme Inhibition - Uncompetitive

• Uncompetitive inhibitors bind only to the ES complex.
• $V_{max}$ is decreased by conversion of ES to ESI which cannot form product.
• Uncompetitive inhibitors reduce [ES].
• As E binds S to replenish ES this apparent increase in affinity of the E for S causes a decrease in $K_m$.
• The Herbicide “Round-Up” is an uncompetitive inhibitor of a plant enzyme involved in amino acid metabolisms.

Reversible Enzyme Inhibition - Non-competitive

• Non-competitive inhibitors bind to E and ES.
• $V<em>{max}$ is decreased with no change in $K</em>m$.
• Non-competitive inhibitors don’t influence S binding, therefor there is no change in $K_m$.
• Essentially reduces the number of active enzyme molecules.
• The antibiotic doxycycline is a non-competitive inhibitor of a bacterial enzyme (collagenase).

Serine Proteases - General Properties

Serine Proteases:

• Serve as digestive enzymes, including trypsin, chymotrypsin, and elastase, that cleave peptide bonds in protein substrates.
• Members of this family share similar sequences and active site residues.
• Are synthesized and stored in the pancreas as inactive zymogens to prevent damage to cellular proteins.
• Zymogens are activated at the appropriate time by selective proteolysis.
• Catalytic mechanism contains elements of both covalent and acid-base catalysis.

Serine Proteases - Substrate Specificities

• Serine proteases have unique specificities that reflect unique substrate binding pockets.
  • Thrombin cleaves Arg-Gly bonds
  • Trypsin cleaves by Lys and Arg.
  • Chymotrypsin cleaves by Phe, Tyr or Met.
  • Elastase cleaves by Gly and Ala.
  • Papain cuts all peptide bonds.

Serine Protease - Catalytic Triad

• Serine Proteases have a conserved catalytic mechanism based on a catalytic triad of residues (Asp, His, Ser)
• Each residue plays a specific role in the catalytic mechanism
  • His acts to accept and donate a proton at each of the two stages of the reaction mechanism (acid base catalysis).
  • Asp stabilizes the positively-charged His to facilitate serine ionization.
  • Ser attacks the carbonyl group of the peptide bond to be cleaved (covalent catalysis).

Chymotrypsin Mechanism - Overview

Phase I

• Step 1: (Acid/Base) Histidine acts as a base to extract proton from hydroxyl of Ser. This activates the oxygen of the hydroxyl group.
• Step 2: (Covalent) Formation of a covalent linkage from the hydroxyl group of the Ser to the carbonyl carbon of the peptide bond to be cleaved in the substrate.
• Step 3: (Acid/Base) Histidine acts as an acid to donate a proton to the amine group of peptide bond to be cleaved, this cuts the substrate peptide into two pieces.

Phase II

• Step 1: (Acid/Base) Histidine acts as a base to extract a proton from a water molecule, activating the oxygen of this molecule.
• Step 2: (Covalent) Activated water molecule attacks the point of covalent linkage between enzyme and substrate.
• Step 3: (Acid/Base) Histidine acts as an acid to donate a proton to reform the hydroxyl group of Ser.

Regulation of Enzyme Activity - Overview

• The activity of an enzyme can be regulated by controlling the amount of the enzyme (long term), or by adjusting the activity of a constant quantity of the enzyme (short term).

1. Regulation of enzyme availability
   • location, rates of synthesis and degradation
2. Regulation of enzyme activity
   • covalent modification -phosphorylation, methylation, glycosylation, etc.
   • non-covalent modification (allosteric) -allosteric regulation

Regulation of Enzyme Activity - Points of Regulation

What would be the logical point to regulate a reaction pathway?

• Enzymatic pathways often controlled through negative feedback inhibition by the final product of the pathway.
• The final product often inhibits the enzyme catalyzing the first unique and committed step.
• Regulation at this step conserves material and energy and prevents accumulation of intermediates.

Regulation of Enzyme Activity - Points of Regulation

• Negative feedback in a branched pathway often occurs by the final product of each branch acting to inhibit the enzyme catalyzing the first unique and committed step of the branch.
  • Enzyme 7 inhibited by G
  • Enzyme 4 inhibited by T

Regulation of Enzyme Activity - Points of Regulation

• Regulation when two pathways cooperate to form a single product.
• The final product can inhibit the first unique step of each branch.
• The molecules preceding the merger can inhibit the first step of their branch as well as activating the first step of the opposing branch.

Allosteric Enzymes - General Properties

Allosteric Enzymes:

• serve as information sensors to coordinate cellular metabolism.
• are regulated by interaction with metabolic intermediates.
• are regulated by allosteric modulators that bind non-covalently at sites other than the active site.
• are usually examples of quaternary structure.
• often catalyze branch-point reactions.
• are often slow, representing the rate limiting step of the pathway.
• do not obey Michaelis-Menten kinetics, instead have sigmoidal curves.

Allosteric Enzymes - General Properties

• Activities of allosteric regulator enzymes are changed by inhibitors and activators (modulators).
• Allosteric modulators bind non-covalently to the enzymes that they regulate.
• Regulatory enzymes often possess quaternary structure.
• There is a rapid transition between the active (R) and inactive (T) conformations.
• Substrates and activators may bind only to the R state while inhibitors may bind only to the T state.
• The binding of the substrate disrupts the R to T equilibrium in favor of R. – This is the basis of the co-operative activation of allosteric enzymes.

Allosteric Enzymes - Physiological Significance of Cooperativity

• Allosteric enzymes transition from a less active state to a more active state within a narrow range of substrate concentration.
• The activity of allosteric enzymes is more sensitive to changes in substrate concentration near the $K<em>m$ than are Michaelis-Menten enzymes of the same $V</em>{max}$.
• This sensitivity is called a threshold effect: below a certain substrate concentration there is little enzyme activity; after the threshold has been reached the enzyme activity increases rapidly (on/off).

Allosteric Enzymes - Phosphofructokinase 1

• PFK1 catalyzes an early step of glycolysis.
• Phosphoenolpyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK1.
• ADP is an allosteric activator of PFK1.
• When the ratio [PEP]/[ADP] is high, PFK1 is inhibited; When the ratio of [PEP]/[ADP] is low, PFK-1 is activated and glycolysis produces more ATP from ADP.
• Thus the concentrations of PEP and ADP act allosterically through PFK1 to regulate the activity of the entire pathway.

Allosteric Enzymes - Activation of Phosphofructokinase-1 by ADP

• The activity of PFK1 is responsive to the concentration of the substrate as well as the allosteric activators and inhibitors.
• Even at constant levels of substrate the activity of the enzyme can be modulated through changes in levels of the allosteric modulators.

Enzyme Regulation by Covalent Modification - General

• Many enzymes are regulated through the covalent linkage of a modifying group to changes some aspect of the proteins behavior, such as activity.
• A number of different types of covalent modification have been characterized (methylation, acetylation, etc).
• The most common post-translation covalent modification is through phosphorylation.
• These modifications are usually reversible with one enzyme catalyzing the addition of the group and another enzyme catalyzing its removal.
• Kinases add phosphoryl groups, phosphatases remove them.

Enzyme Regulation by Covalent Modification - Glycogen Metabolism

• Production and utilization of glycogen is controlled by two enzymes:
  • glycogen synthase (anabolic) which catalyzes production of glycogen from glucose.
  • glycogen phosphorylase (catabolic) which catalyzes the breakdown of glycogen into glucose.

Enzyme Regulation by Covalent Modification - Glycogen Metabolism

• In response to hormones that are released when you are hungry (glucagon) or scared (epinephrine) both enzymes are phosphorylated
• Phosphorylation activates the catabolic enzyme and inactivates the anabolic enzyme.
• This situation favors the breakdown of glycogen into glucose.

Enzyme Regulation by Covalent Modification - Glycogen Metabolism

• In response to hormones released in the fed state (insulin) both enzymes are unphosphorylated.
• When unphosphorylated the anabolic enzyme is active and the catabolic enzyme is inactive.
• This situation favors the storage of glucose within glycogen.