Chapter 6 enzymes
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Lehninger Principles of Biochemistry, Fourth Edition, 2005. Chapter 6 Enzymes. 中央研究院 生物化學研究所 曾湘文 博士 Mar. 27, 2007 . There are two fundamental conditions for life:. First, the living entity must be able to self-replicate ;

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Chapter 6 Enzymes

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Chapter 6 enzymes

Lehninger Principles of Biochemistry, Fourth Edition, 2005

Chapter 6Enzymes



曾湘文 博士

Mar. 27, 2007

There are two fundamental conditions for life

There are two fundamental conditions for life:

  • First, the living entity must be able to self-replicate;

  • second, the organism must be able to catalyzechemical reactions efficiently and selectively.



6.1 An Introduction to Enzymes

6.2 How Enzymes Work

6.3 Enzyme Kinetics as an Approach to Understanding Mechanism

6.4 Examples of Enzymatic Reactions

6.5 Regulatory Enzymes

Chapter 6 enzymes

Introduction to Enzymes

1897 Eduard Buchner --- yeast extracts can ferment sugar to alcohol

Frederick W. Kuhne --- the name “enzyme”

1926 James Sumner --- crystallization of urease

John Northrop & Moses Kunitz --- crystallization of pepsin and trypsin

J.B.S. Haldane --- treatise for “Enzymes” (weak-bonding interactions)

Most enzymes are proteins

Most enzymes are proteins

Most Enzymes Are Proteins

  • Catalytic RNA (exception) = Ribozyme

  • An enzyme is denatured or dissociated into subunits, catalytic activity is usually lost

  • The primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their catalytic activity.

  • Some enzymes require no chemical groups for activity other than their amino acid residues. Others require an additional chemical component called a cofactor(inorganic ions) orcoenzyme (complex organic or metalloorganic molecule).

Chapter 6 enzymes

Holoenzyme (全酶) = Apoenzyme + Coenzyme or Cofactor

Coenzymes and cofactors holoenzyme apoenzyme coenzyme or cofactor

Coenzymes and Cofactors (Holoenzyme = apoenzyme + coenzyme or cofactor)

  • Cofactors: additional chemical component inorganic ions: Fe2+, Mg2+, Mn2+ or Zn2+.

  • Coenzyme: complex organic or metallorganic molecule, transient carriers of specific function groups.

  • Prosthetic group: a coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein.

  • Holoenzyme : A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions)

  • Apoenzyme (apoprotein): protein part of holoenzyme

Nomenclature of enzymes classified by the reactions they catalyze

Nomenclature of enzymes: Classified by the Reactions They Catalyze

  • Suffix (~ase) to the name of their substrate or to a word or phrase describing their activity. Ex. Urease: hydrolysis of urea

  • Enzyme Commission number (E.C.): divides enzymes into six classes, each with subclasses, based on the type of reaction catalyzed.

Nc iubmb system four part classification number of enzyme

NC-IUBMB system: Four-part classification number of enzyme

  • Hexokinase (ATP: glucose phosphotransferase; E.C. (class. subclass. phosphotransferase with a hydorxyl as acceptor. D-glucose as receptor).

  • The first number (2) denotes the class name (transferase);

  • The second number (7), the subclass (phosphotransferase);

  • The third number (1), the subsubclass ; a phosphotransferase with a hydroxyl group as acceptor;

  • The fourth number (1), D-glucose as the phosphoryl group acceptor.

Nomenclature Committee of the International Union of Biochemistry and Molecular Biology :

6 2 how enzymes work

6-2 How Enzymes Work?

  • Binding of a substrate to an enzyme at the active site.

  • The enzyme chymotrypsin, with bound substrate in red.

  • Some key active-site amino acid residues appear as a red splotch on the enzyme surface.

Enzymes affect reaction rates not equilibria

Enzymes Affect Reaction Rates, Not Equilibria

  • E, S, and P represent the enzyme, substrate, and product;

  • ES and EP are transient complexes of the enzyme with the substrate and with the product.

Reaction coordinate diagram for a chemical reaction

Reaction coordinate diagram for a chemical reaction

  • The free energy (G) of the system is plotted against the progress of the reaction S→P.

  • The horizontal axis (reaction coordinate) reflects the progressive chemical changes (e.g., bond breakage or formation) as S is converted to P.

  • The G‡ (activation energies) for the S → P and P → S reactions are indicated.

  • DG 0is the change of standard free-energy (298 K; 1 atm, or 101.3 kPa; 1 M) in the direction S → P.

  • DG ’0 is the biochemical standard free-energy change (standard free-energy change at pH 7.0) .

Reaction coordinate diagram comparing enzyme catalyzed and uncatalyzed reactions

Reaction coordinate diagram comparing enzyme-catalyzed and uncatalyzed reactions

  • Enzymes affect reaction rates, Not equilibria, by lowering activation energies.

  • The free E of the ground state of P is lower than that of S, so DG’o, for the reaction is negative and the equilibrium favors P. The position and direction of equilibrium are not affect by any catalyst.

  • A higher activation energy (DG‡) corresponds to a slower reaction: can be increased by raising temp. or adding catalyst.

  • Rate-limiting step: step with highest activation E.

  • The ES and EP intermediates occupy minima in the energy progress curve of the enzyme-catalyzed reaction.

Reaction rates and equilibria have precise thermodynamic definitions

Reaction Rates and Equilibria have precise thermodynamic definitions

  • A large negative value for DG ’0 reflects a favorable reaction equilibria—but this does not mean the reaction will proceed at a rapid rate.

  • A lower activation energy means a higher reaction rate.

  • Equilibrium constant (Keqor K )

  • Ris the gas constant, 8.315 J/mol K, and T is the absolute temperature, 298 K (25 C).

  • Rate equation (V):

    V =k [S] –first-order

    V =k [S1] [S2] -second-order

Catalytic power and specificity of enzymes

Catalytic Power and Specificity of Enzymes

  • The rate enhancements they bring about are in the range of 5 to 17 orders of magnitude.

  • What is the source of the energy for the dramatic lowering of the activation energies for specific reactions?

    • The first lies in the rearrangements of covalent bonds during an enzyme-catalyzed reaction.

    • The second part of the explanation lies in the noncovalentinteractions between enzyme and substrate.

Chapter 6 enzymes

Two fundamental and interrelated principles provide a general explanation for how enzymes use noncovalent binding energy:

  • Much of the catalytic power of enzymes is ultimately derived from the free energy released in forming many weak bonds and interactions between an enzyme and its substrate. This binding energy contributes to specificity as well as to catalysis.

  • Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrates per se but to the transition states through which substrates pass as they are converted to products during an enzymatic reaction.

Weak interactions between enzyme and substrate are optimized in the transition state

Weak interactions between enzyme and substrate are optimized in the transition state

  • Catalytic functional group forms transient covalent bond with substrate (lower the reaction rate).

  • Formation of noncovalent interactions between enzyme and substrate ---release of free E that provide a degree of stabilization.

  • DGB (binding energy): the E derived from enzyme-substrate interaction--- major source of free energy used by enzymes to lower the activation energies of reactions.

  • Free E released in forming weak bonds: Bind E contributes to specificity as well as catalysis.

  • Enzyme active sites are complementary not to the substrates; but to transition states (intermediate)

Complementary shapes of a substrate and its binding site on an enzyme

Complementary shapes of a substrate and its binding site on an enzyme

  • The enzyme dihydrofolate reductase with its substrate NADP (red), unbound (left) and bound (right). Another bound substrate, tetrahydrofolate (yellow).

  • The NADP binds to a pocket that is complementary to it in shape and ionic properties.

  • The interaction of a protein with a ligand often involves changes in the conformation of one or both molecules, a process called induced fit.

  • This lack of perfect complementarity between enzyme and substrate is important to enzymatic catalysis.

An imaginary enzyme stickase designed to catalyze the breaking of a metal stick

An imaginary enzyme (stickase) designed to catalyze the breaking of a metal stick

  • The binding energy of the magnetic interactions compensates for the increase in free energy required to bend the stick.

  • When the enzyme is complementary to the substrate, the ES complex is more stable and has less free energy in the ground state than substrate alone, The result is an increasein the activation energy.

Role of binding energy in catalysis

Role of binding energy in catalysis

  • To lower the activation energy for a reaction, the system must acquire an amount of energy equivalent to the amount by which △G‡ is lowered.

  • Much of this energy comes from binding energy (DGB) contributed by formation of weak noncovalent interactions between substrate and enzyme in the transition state.

Specific catalytic groups contribute to catalysis

Specific Catalytic Groups Contribute to Catalysis

  • Once a substrate is bound to an enzyme, properly positioned catalytic functional groups aid in the cleavage and formation of bonds by a variety of mechanisms, including:

    • General acid-base catalysis,

    • Covalent catalysis,

    • Metal ion catalysis.

General acid base catalysis

General acid-base catalysis

  • Charged intermediates can often be stabilized by the transfer of protons to or from the substrate or intermediate to form a species that breaks down more readily to products.

  • For nonenzymatic reactions, the proton transfers can involve either the constituents of water alone or other weak proton donors or acceptors.

  • Catalysis of this type that uses only the H (H3O) or OH ions present in water is referred to as specific acid-base catalysis.

  • Stabilized by transfer of protons to or from the substrate or intermediate to form a breaks down more readily to products than to reactants.

Amino acids in general acid base catalysis

Amino acids in general acid-base catalysis

  • Many organic reactions are promoted by proton donors (general acids) or proton acceptors (general bases).

  • The active sites of some enzymes contain amino acid functional groups, that can participate in the catalytic process as proton donors or proton acceptors.

Covalent and general acid base catalysis chymotrypsin

Covalent and general acid-base catalysis (chymotrypsin)

  • Most enzymes employ a combination of several catalytic strategies to bring about a rate enhancement.

  • Chymotrypsin - both covalent catalysis and general acid-base catalysis:

    • cleavage of a peptide bond accompanied by formation of a covalent linkage between a Ser residue on the enzyme and part A transient covalent bond is formed between the enzyme and the substrate.

    • the reaction is enhanced by general base catalysis by other groups on the enzyme

  • Ions help orient the substrate; stabilized charged reaction transition states; oxidation-reduction reaction.

6 3 enzyme kinetics as an approach to understanding mechanism

6.3 Enzyme Kinetics as an Approach to Understanding Mechanism

Enzyme kinetics: determination of the rate of the reaction and how it changes in response to changes in experimental parameters

Effect of substrate

Concentration on the initial

velocity of an

enzyme-catalyzed reaction

V0 (initial velocity)

when [S]>>[E], t is short

Vmax (maximum velocity)

when [S] 

Effect of substrate concentration on the initial velocity of an enzyme catalyzed reaction

Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction

  • Vmax is extrapolated from the plot, because V0 approaches but never quite reaches Vmax.

  • The substrate concentration at which V0 is half maximal is Km, the Michaelis constant.

  • The concentration of enzyme in an experiment such as this is generally so low that [S] >> [E] even when [S] is described as low or relatively low.

  • The units shown are typical for enzyme-catalyzed reactions and are given only to help illustrate the meaning of V0 and [S].

Steady state kinetics michaelis menten equation



Steady-state kinetics: Michaelis-Menten equation



Formation of ES=k1 [E][S] = k1([Et]-[ES])[S] because [E] = ([Et]-[ES])

Breakdown of ES=k-1[ES]+k2[ES]

K1 [Et] [S] - k1[ES] [S] = (k-1 + k2) [ES]

K1 [Et] [S] = ( k1 [S] + k-1 + k2) [ES]

[ES] = [Et] [S] / [S] +(k2+k-1)/k1

[ES]= [Et] [S] / [S] + km

Vo = k2 {[Et] [S] / [S] + km} because Vo =k2 [ES]

= Vmax [S] / {[S] + km}

Vo= ½ Vmax(when Km= [S], unit = mM)

Michaelis constant:

Chapter 6 enzymes

Michaelis-Menten equation

When [S] = Km V0 = ½ Vmax

Lineweaver burk equation double reciprocal plot transformation of the michaelis menten equation

Lineweaver-burk equation (Double-Reciprocal Plot):transformation of the Michaelis-Menten equation

K m and v max

Km and Vmax

  • K-1>>k2 ---- Kd of ES (disassocaition Constance)

  • But when K-1<<K2, Km = k2/k1

  • If an enzyme reacts by the two-step Michaelis-Menten mechanism, Vmax = k2[Et], where k2 is rate-limiting. However, the number of reaction steps and the identity of the rate-limiting step(s) can vary from enzyme to enzyme.

  • Early in the reaction [P] is very low: Vmax = k3[Et].

  • Turnover number (kcat): number of substrate molecules converted to product in a given unit of time (S-1).

  • Specificity constant (kcat/Km): compare the catalytic efficiencies of different enzymes or the turnover of different substrate by same enzyme.

Many enzymes catalyze reactions with two or more substrates

Many enzymes catalyze reactions with two or more substrates

  • The enzyme and both substrates come together to form a ternary complex. In ordered binding, substrate 1 must bind before substrate 2 can bind productively.

  • In random binding, the substrates can bind in either order.

Ping pong or double displacement mechanism

Ping-Pong, or double displacement, mechanism

  • An enzyme-substrate complex forms, a product leaves the complex, the altered enzyme forms a second complex with another substrate molecule, and the second product leaves, regenerating the enzyme.

  • Substrate 1 may transfer a functional group to the enzyme (to form the covalently modified E), which is subsequently transferred to substrate.

  • No ternary complex is formed

Steady state kinetic analysis of bisubstrate reactions

Steady-state kinetic analysis of bisubstrate reactions

  • The concentration of substrate 1 is varied while the concentration of substrate 2 is held constant.

  • This is repeated for several values of [S2], generating several separate lines.

  • (a) Intersecting lines indicate that a ternary complex is formed in the reaction;

  • (b) parallel lines indicate a Ping-Pong (double-displacement) pathway.



Enzymes are subject to reversible or irreversible inhibition

Enzymes Are Subject to Reversible or Irreversible Inhibition

  • Reversible Inhibition

    • Competitive inhibition

    • Uncompetitive inhibition

    • Mixed inhibition

  • Irreversible Inhibition

Chapter 6 enzymes

  • A competitive inhibitor (resemble the substrate) competes with the substrate for the active site of an enzyme. While the inhibitor (I) occupies the active site it prevents binding of the substrate to the enzyme.

  • When [S] >> [I], the probability that an inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax.

Chapter 6 enzymes

  • An uncompetitive inhibitor binds at a site distinct from the substrate active site and, binds only to the ES complex.

  • An uncompetitive inhibitor lowers the measured Vmax. Apparent Km also decreases.

Chapter 6 enzymes

  • A mixed inhibitor also binds at a site distinct from the substrate active site, but it binds to either E or ES. The rate equation describing mixed inhibition

  • Noncompetitive inhibition (special case- rarely encountered in experiments) : a = a’ --- affect the Vmax but not the Km.

Apparent v max and k m

uncompetitive and mixed inhibition are observed only for enzymes with two or more substrates — S1 and S2

Apparent Vmax and Km

Irreversible inhibitor

Irreversible inhibitor

  • The irreversible inhibitors: 1). bind covalently with or destroy a functional group on an enzyme; 2). form a particularly stable noncovalent association.

  • Suicide inactivators (mechanism-based inactivators) :undergoes the first few chemical steps of the normal enzymatic reaction, but instead of being transformed into the normal product - converted to a very reactive compound that combines irreversibly with the enzyme

  • highjack the normal enzyme reaction mechanism to inactivate the enzyme. Suicide inactivators play a significant role in rational drug design --- few side effects

  • Fig. Reaction of chymotrypsin with diisopropylfluorophosphate (DIFP) irreversibly inhibits the enzyme. This has led to the conclusion that Ser195 is the key active-site Ser residue in chymotrypsin.

Enzyme activity is affected by ph

Enzyme activity is affected by pH

  • Amino acid side chains in the active site may act as weak acids and bases with critical functions that depend on their maintaining a certain state of ionization.

  • in the protein ionized side chains may play an essential role in the interactions that maintain protein structure.

  • Ex. Removing a proton from a His residue, - might eliminate an ionic interaction essential for stabilizing the active conformation of the enzyme. A less common cause of pH sensitivity is titration of a group on the substrate.

  • Caution - A nearby positive charge can lower the pKa of a Lys residue, and a nearby negative charge can increase it.

6 4 examples of enzymatic reactions

6.4 Examples of Enzymatic Reactions

  • The Chymotrypsin Mechanism Involves Acylation and Deacylation of a Ser Residue

  • Hexokinase Undergoes Induced Fit on Substrate Binding

  • The Enolase Reaction Mechanism Requires Metal Ions

  • Lysozyme Uses Two Successive Nucleophilic Displacement Reactions

Structure of chymotrypsin

Structure of chymotrypsin

  • Chymotrypsin is a serine protease with a well-understood mechanism, featuring general acid-base catalysis, covalent catalysis, and transition-state stabilization.

Hydrolytic cleavage of a peptide bond by chymotrypsin

Hydrolytic cleavage of a peptide bond by chymotrypsin

Induced fit in hexokinase

Induced fit in hexokinase

  • Hexokinase has a U-shoped structure and provides an excellent example of induced fit as a means of using substrate binding energy.

  • The ends pinch toward each other in a conformational change induced by binding of D-glucose (red)

  • side chains out of position in an inactive conformation glucose (but not water) and Mg ATP bind, the binding energy derived from this interaction induces a conformational change.

  • Xylose binds to hexokinase -cannot be phosphorylated. But ATP hydrolysis.

Two step reaction catalyzed by enolase require metal ions

Two-step reaction catalyzed by enolase (require metal ions)

  • The carboxyl group of 2-PGA is coordinated by two magnesium ions at the active site. A proton is abstracted in step 1 by general base catalysis (Lys345), and the resulting enolic intermediate is stabilized by the two Mg 2+ ions.

  • Elimination of the -OH in step 2 is facilitated by general acid catalysis (Glu211).

  • The substrate, 2-PGA, in relation to the Mg2+ ions, Lys345, and Glu211 in the enolase active site. All the oxygen atoms of 2-PGA are light blue; phosphorus is orange.

Chapter 6 enzymes

Hen egg white lysozyme and the reaction it catalyzes

  • Lysozyme makes use of covalent catalysis and general acid catalysis as it promotes two successive nucleophilic displacement reactions.

6 5 regulatory enzymes

6.5 Regulatory Enzymes

Chapter 6 enzymes

6.5 Regulatory EnzymesAllosteric enzymes undergo conformational changes in response to modulator binding

  • In many allosteric enzymes the substrate binding site and the modulator binding site(s) are on different subunits, the catalytic (C) and regulatory (R) subunits, respectively.

  • Binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change.

  • This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On dissociation of the modulator from the regulatory

  • subunit, the enzyme reverts to its inactive or less active form.

The regulatory step in many pathways is catalyzed by an allosteric enzyme feedback inhibitions

The regulatory step in many pathways is catalyzed by an allosteric enzyme --- Feedback inhibitions

  • The regulatory enzyme is specifically inhibited by the end product of the pathway whenever the concentration of the end product exceeds the cell’s requirements.

  • When the regulatory enzyme reaction is slowed, all subsequent enzymes operate at reduced rates as their substrates are depleted.

  • Usual at the first step or branch

The kinetic properties of allosteric enzymes diverge from michaelis menten behavior

The kinetic properties of allosteric enzymes diverge from Michaelis-Menten behavior

  • Substrate-activity curves for representative allosteric enzymes.

  • (a) The sigmoid curve of a homotropic enzyme, in which the substrate also serves as a positive (stimulatory) modulator, or activator. Note the resemblance to the oxygen-saturation curve of hemoglobin.

  • (b) The effects of a positive modulator (+) and a negative modulator (-) on an allosteric enzyme in which K0.5 is altered without a change in Vmax. The central curve shows the substrate-activity relationship without a modulator.

  • (c) A less common type of modulation, in which Vmax is altered and K0.5 is nearly constant.

Reversible covalent modification

Reversible covalent modification

  • Modifying groups include phosphoryl, adenylyl, uridylyl, methyl, and adenosine diphosphate ribosyl groups - linked to and removed by separate enzymes.

Regulation of glycogen phosphorylase activity by phosphorylation

Regulation of glycogen phosphorylase activity by phosphorylation

  • Phosphoryl groups affect the structure and catalytic activity of proteins (Kinase Vs.phosphatases)

  • Phosphoryl group on S, T and Y--- charge (polar), P-O forms H bonds, electric repulsion (Asp and Glu). --- conformation change to more active from or change substrate binding affinity

Multiple phosphorylations allow exquisite regulatory control glycogen synthase

Multiple phosphorylations allow exquisite regulatory control --- Glycogen synthase

  • Consensus sequence

  • Hierarchical phosphorylation -- a certain residue can be phosphorylated only if a neighboring residue has been phosphorylated first.

  • The glycogen synthase contains at least 9 sites in five designated regions susceptible to phosphorylation by one of the cellular protein kinase. The activity of this enzyme is therefore capable of modulation in response to a variety of signals. Thus regulation is a matter not of binary switch but of finely tuned modulation of activity over a wide range.

Activation of zymogens by photolytic cleavage

Activation of zymogens by photolytic cleavage

  • Zymogen: an inactive precursor of the enzyme (e.g. chymotrpsin—chymotrypsinogen and trypsin—trypsinogen)

  • Specific cleavage causes conformational change –expose active site. (irreversible) -- proteases inhibitors bind very tightly to the enzyme active site (e.g. pancreatic trypsin inhibitor– trypsin; a1-antiproteinase – neutrophil elastase.

  • Proproteins or proenzymes, e.g. fibrin (from fibrinogen); thrombin (from prothrombin).

Protein ligand interaction

Protein-Ligand interaction

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