Enzymes Basic Concepts and Kinetics 79

Enzymes

2009

Biochemistry

Pre professional year

Dr. Waleed Tamimi

Outline1. Enzymes are powerful and highly specific catalysts.

2. Free energy is a useful Thermodynamic function for understanding Enzymes.

3. Enzymes accelerate Reactions by facilitating the formation of the transition State.

4. The Mechaelis-Menten formula accounts for the kinetics properties of many enzymes.

5. Enzymes can be inhibited by specific molecules.

6. Vitamins are often precursors to Coenzymes.

Definition & Function

• Characteristics of enzymes are their catalysis and specificity.

• Catalysis takes place at a particular site on the enzyme called the active site.

• Nearly all known enzymes are proteins.

Functions of Enzymes Are Powerful and Highly Specific Catalysts

• Are Powerful and Highly Specific Catalysts• Enzymes accelerate reactions by factors of as much as a million

or more.• Biological reactions do not take place in absence of enzymes. • Example is the hydration of carbon dioxide is catalyzed by an

enzyme—carbonic anhydrase.• The transfer of CO2 from the tissues into the blood and then to

the alveolar air would be less complete in the absence of this enzyme.

Proteolytic Enzymes

• Proteolytic Enzymes catalyze proteolysis, the hydrolysis of a peptide bond.

• The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme.

Examples of Proteolytic Enzymes • Subtilisin is found in bacteria, it cleaves any

peptide bond.

• Trypsin, a digestive enzyme, catalyzes the splitting of peptide bonds only on the carboxyl side of lysine and arginine residues.

• Thrombin, an enzyme that participates in blood clotting, catalyzes the hydrolysis of Arg-Gly bonds.

• DNA polymerase I, adds nucleotides to a DNA strand.

Figure 8.1. Enzyme Specificity. (A) Trypsin cleaves on the carboxyl side of arginine and lysine residues, whereas (B) Thrombin cleaves Arg-Gly bonds in particular sequences specifically.

Enzymes Are Classified on the Basis of the Types of Reactions That They Catalyze

• Many enzymes have common names that provide little information about the reactions that they catalyze (e.g., trypsin).

• Most other enzymes are named for their substrates and for the reactions that they catalyze, with the suffix “ase” added.

• Thus, an ATPase is an enzyme that breaks down ATP, while ATP synthase is an enzyme that synthesizes ATP.

An Energy-Transforming Enzyme. Ca2+ ATPase uses the energy of ATP hydrolysis to transport Ca2+ across the membrane, generating a Ca2+ gradient.

Using of ATP

• The enzyme myosin converts the energy of ATP into the mechanical energy of contracting muscles.

• Pumps in the membranes of cells, which can be thought of as enzymes that move substrates, create chemical and electrical gradients by using the energy of ATP to transport molecules and ions

Enzyme nomenclature

• 1964 the International Union of Biochemistry established an Enzyme Commission to develop a nomenclature for enzymes.

• Reactions were divided into six major groups numbered 1 through 6. These groups were subdivided and further subdivided, so that a four-digit number preceded by the letters EC for Enzyme Commission could precisely identify all enzymes.

• Nucleoside Monophosphate (NMP) kinase EC 2.7.4.4.

Six major classes of enzymes

Active site• A specific enzyme catalyzes each cellular reaction.• A unique 3-D shape of an enzyme determines

which chemical reaction it catalyzes.• A specific reactant that an enzyme acts on is called

a substrate of the enzyme.• A substrate fits into a region of the enzyme called

an active site.• An active site is typically a pocket or groove on

the surface of the enzyme.

Substrate

Active site

Enzyme Enzyme-substratecomplex

When a substrate binds to an enzyme, the active site changes shape slightly so that it embraces the substrate more snugly – induced fit.

How Enzymes work?• Enzymes speed up the cell’s chemical reactions by

lowering energy barriers.• There is an energy barrier that must be overcome before

a chemical reaction can begin – the energy of activation (EA).

• The reactants must absorb EA to become activated – to contort or weaken bonds so that they can break and new bonds can form, and start a reaction.

• An enzyme is a protein molecule that functions as a biological catalyst.

• The enzyme increases the rate of a reaction without itself being changed.

An enzyme speeds up a reaction by lowering the EA barrier.

Each reaction has a transition state where the substrate is in an unstable, short-lived chemical/structural state.

Enzymes act by lowering the freeenergy of the transition state

How Enzymes work?

• Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate

• The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction

• Not a lock & key but induced fit – the enzyme and/or the substrate distort towards the transition state

Enzymes Decrease the Activation Energy. Enzymes accelerate reactions by decreasing ΔG‡, the free energy of activation.

The Formation of an Enzyme-Substrate Complex Is the

First Step in Enzymatic Catalysis • The first step in catalysis is the formation of an enzyme-

substrate complex.The substrates are bound to a specific region of the enzyme called the active site.

• The recognition of substrates by enzymes is accompanied by conformational changes at active sites, and such changes facilitate the formation of the transition state.

Reaction Velocity Versus Substrate Concentration in an Enzyme-Catalyzed Reaction. An enzyme-catalyzed reaction reaches a maximal velocity.

Common Features of Active Sites of Enzymes

1. The active site is a three-dimensional cleft

2. The active site takes up a relatively small part of the total volume of an enzyme.

3. Active sites are clefts or crevices.

4. Substrates are bound to enzymes by multiple weak attractions

5. The specificity of binding depends on the precisely defined arrangement of atoms in an active site. lock and key or induced fit

Lock-and-Key Model of Enzyme-Substrate Binding. In this model, the active site of the unbound enzyme is complementary in shape to the substrate.

Induced-Fit Model of Enzyme-Substrate Binding. The enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound.

Part 2

Enzyme Kinetics and Inhibition

The Michaelis-Menten Model Accounts for

the Kinetic Properties of Many Enzymes

• To understand how enzymes function, we need a kinetic description of their activity.

• The Michaelis-Menten equation relates the initial velocity of an enzyme-catalyzed reaction, Vi, to the concentration of substrate, S, and two parameters, Km and Vmax.

• Vmax is the velocity of the reaction extrapolated to infinite substrate concentration and Km is the substrate concentration at which the initial velocity equals ½ Vmax.

Michaelis-Menten Kinetics. A plot of the reaction velocity (V0) as a

function of the substrate concentration [S] for an enzyme that obeys Michaelis-Menten kinetics shows that the maximal velocity (Vmax) is

approached asymptotically. The Michaelis constant (KM) is the substrate

concentration yielding a velocity of Vmax/2.

Michaelis-Menten

• In 1913, Leonor Michaelis and Maud Menten proposed a simple model to account for these kinetic characteristics.

• The ES complex has two possible fates. – It can dissociate to E and S, with a rate constant k-1, – or it can proceed to form product P, with a rate

constant k2.

Michaelis-Menten

• the catalytic rate is equal to the product of the concentration of the ES complex and k2.

• when [S] is much less than KM, then V0 = (Vmax/KM)[S];

that is, the rate is directly proportional to the substrate concentration.

• when [S] is much greater than KM, V0 = Vmax; that is, the

rate is maximal, independent of substrate concentration.

• When [S] = KM, then V0 = Vmax/2.

• Thus, KMis equal to the substrate concentration at which

the reaction rate is half its maximal value.

• KM is an important characteristic of an enzyme-catalyzed

reaction and is significant for its biological function.

Michaelis-Menten

The physiological consequence of KM

• It is illustrated by the sensitivity of some individuals to ethanol.

• Such persons exhibit facial flushing and rapid heart rate (tachycardia) after ingesting even small amounts of alcohol.

• In the liver, alcohol dehydrogenase converts ethanol into acetaldehyde.

The physiological consequence of KM

• Acetaldehyde, is processed to acetate by acetaldehyde dehydrogenase.

• Most people have two forms of the acetaldehyde dehydrogenase, a low KM mitochondrial form and a high KM cytosolic form.

• In susceptible persons, the mitochondrial enzyme is less active due to the substitution of a single amino acid, and acetaldehyde is processed only by the cytosolic enzyme.

• Because this enzyme has a high KM, less acetaldehyde is converted into acetate; excess acetaldehyde escapes into the blood and accounts for the physiological effects.

Kinetic Perfection in Enzymatic Catalysis

• The kcat/KM Criterion. When the substrate concentration is much greater than KM, the rate of catalysis is equal to kcat, the turnover number

• Most Biochemical Reactions Include Multiple Substrates

• Allosteric Enzymes (display sigmoidal plots in which the binding of substrate becomes cooperative ) Do Not Obey Michaelis-Menten Kinetics

• cooperativity results in a sigmoidal plot of V0 versus [S].

Kinetics for an Allosteric Enzyme. Allosteric enzymes display a sigmoidal dependence of reaction velocity on substrate concentration.

Enzymes Can Be Inhibited by Specific Molecules

• Specific small molecules, ions, drugs and toxic agents act by inhibiting enzymes

• Enzyme inhibition can be either reversible or irreversible.

Irreversible inhibitor• An irreversible inhibitor dissociates very slowly

from its target enzyme because it is tightly bound to the enzyme, either covalently or noncovalently.

• Penicillin acts by covalently modifying the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls and thus killing the bacteria.

• Aspirin acts by covalently modifying the enzyme cyclooxygenase, reducing the synthesis of inflammatory signals.

Reversible inhibition• Reversible inhibition, in contrast with irreversible

inhibition, is characterized by a rapid dissociation of the enzyme-inhibitor complex.

• In competitive reversible inhibition, an enzyme can bind substrate (forming an ES complex) or inhibitor (EI) but not both (ESI).

• The competitive inhibitor resembles the substrate and binds to the active site of the enzyme.

• The substrate is thereby prevented from binding to the same active site.

• A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.

Distinction between a Competitive and a Noncompetitive Inhibitor. (Top) enzyme-substrate complex; (middle) a competitive inhibitor binds at the active site and thus prevents the substrate from binding; (bottom) a noncompetitive inhibitor does not prevent the substrate from binding.

Methotrexate

• Methotrexate is a structural analog of tetrahydrofolate, a coenzyme for the enzyme dihydrofolate reductase, which plays a role in the biosynthesis of purines and pyrimidines.

• It binds to dihydrofolate reductase 1000-fold more tightly than the natural substrate and inhibits nucleotide base synthesis.

• It is used to treat cancer.

Figure 8.16. Enzyme Inhibitors. The cofactor tetrahydrofolate and its structural analog methotrexate. Regions with structural differences are shown in red.

• In noncompetitive reversible inhibition, the inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites.

• A noncompetitive inhibitor acts by decreasing the turnover number rather than by diminishing the proportion of enzyme molecules that are bound to substrate.

• Noncompetitive inhibition, in contrast with competitive inhibition, cannot be overcome by increasing the substrate concentration.

• A more complex pattern, called mixed inhibition, is produced when a single inhibitor both hinders the binding of substrate and decreases the turnover number of the enzyme.

Competitive and Noncompetitive Inhibition Are

Kinetically Distinguishable • In competitive inhibition, the inhibitor competes

with the substrate for the active site. • Because increasing the amount of substrate can

overcome the inhibition, Vmax can be attained in the presence of a competitive inhibitor.

• The hallmark of competitive inhibition is that it can be overcome by a sufficiently high concentration of substrate.

• However, the apparent value of KM is altered; the effect of a competitive inhibitor is to increase the apparent value of KM

Competitive and Noncompetitive Inhibition

Are Kinetically Distinguishable

• Competitive increase the Km of the enzyme, but not the Vmax.

• A noncompetitive inhibitor will change the Vmax of the enzyme.

• Uncompetitive inhibitors decrease both Km and Vmax. An uncompetitive inhibitor binds only to the enzyme-substrate complex.

Lineweaver-Burk Transformation

• The Km and Vmax for an enzyme can be visually determined from a plot of 1/v versus 1/S, called a lineweaver-Burk or a double reciprocal plot.

1 = Km 1 + 1

Vi Vmax [S] Vmax

Enzyme Inhibition by Diisopropylphosphofluoridate (DIPF). 1) DIPF can inhibit an chymotrypsin by covalently modifying a crucial serine residue. DIPF modifies only 1 of the 28 serine residues in chymotrypsin. 2) DIPF also revealed a reactive serine residue in acetylcholinesterase, an enzyme important in the transmission of nerve impulses. Thus, DIPF and similar compounds that bind and inactivate acetylcholinesterase are potent nerve gases.

Summary 1) Enzymes are Powerful and Highly Specific

Catalysts

2) Free Energy Is a Useful Thermodynamic Function for Understanding Enzymes

3) Enzymes Accelerate Reactions by Facilitating the Formation of the Transition State

4) The Michaelis-Menten Model Accounts for the Kinetic Properties of Many Enzymes

5) Enzymes Can Be Inhibited by Specific Molecules

6) Vitamins Are Often Precursors to Coenzymes

Part 3

Enzymes Cofactors

• Many enzymes require cofactors to be more active.

• Enzyme without its cofactor is referred to as an apoenzyme; the complete, catalytically active enzyme is called a holoenzyme.

• Cofactors can be subdivided into two groups:

1) metals (coenzymes),derived from vitamins, either tightly or loosely bound to the enzyme.

2) small organic molecules

Vitamins Are Often Precursors to Coenzymes

• Water-Soluble Vitamins Function As Coenzymes

• Ascorbate, the ionized form of ascorbic acid, serves as a reducing agent (an antioxidant), as will be discussed shortly. The vitamin B series comprises components of coenzymes

• Vitamin deficiencies are capable of causing a variety of pathological conditions

Structures of Some Water-Soluble Vitamins.

Structures of Some Fat-Soluble Vitamins

Factors Affecting EnzymeAction: Temperature

• Little activity at low temperature

• Rate increases with temperature

• Most active at optimum temperatures (usually 37°C in humans)

• Activity lost with denaturation at high temperatures

Factors Affecting EnzymeAction: Substrate Concentration

• Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant)

• Maximum activity reached when all of enzyme combines with substrate

Factors Affecting EnzymeAction: pH

• Maximum activity at optimum pH

• R groups of amino acids have proper charge

• Tertiary structure of enzyme is correct

• Narrow range of activity

• Most lose activity in low or high pH

• The cellular environment affects enzyme activity.• An enzyme is most effective under an appropriate

condition.• Temperature affects molecular motion – an enzyme’s

optimal temperature produces the highest rate.• Most human enzymes work best at 35-40 ºC.• Salt concentration and pH influence enzyme activity.• The salt ions interfere with some of the chemical bonds

that maintain protein structure. The same is true of the extra hydrogen ions at very low pH.

• The optimal pH for most enzymes is near neutrality.

• Allosteric regulation controls an enzyme’s activity.

• Allosteric regulation is the term used to describe cases where a protein’s function at one site is affected by binding of a regulatory molecule at another site.

• Allosteric regulation may either inhibit or stimulate an enzyme’s activity by changing an enzyme into its active or inactive forms.

• Not all vitamins function as coenzymes. The fat-soluble vitamins, which are designated by the letters A, D, E, and K , have a diverse array of functions.

• Vitamin K, which is required for normal blood clotting, participates in the carboxylation of glutamate residues to γ-carboxyglutamate, which makes modified glutamic acid a much stronger chelator of Ca2+.

• Vitamin A (retinol) is the precursor of retinal, the light-sensitive group in rhodopsin and other visual pigments.

• A deficiency of this vitamin leads to night blindness.

Fat-Soluble Vitamins

• Vitamin D is regulates the metabolism of calcium and phosphorus. A deficiency in vitamin D impairs bone formation.

• Infertility in rats is a consequence of vitamin E (α-tocopherol) deficiency. This vitamin reacts with and neutralizes reactive oxygen species such as hydroxyl, radicals before they can oxidize unsaturated membrane lipids, damaging cell structures.