Biochemistry SSheng Zhao ( 赵晟 ), Biochemistry and Molecular Department of Medical school in Southeast University WWeb: E or QQQ /MSN/Skype/gChat: MMobile: or Conception, theory, research, and application ——Logic and LIY (Learn It Yourself)

Chapter 3: Enzyme Thermodynamics and Kinetics

Section One The Molecular Structure and Function of Enzyme

Enzymes / ˈɛ nza ɪ mz/ are large biological molecules responsible for the thousands of chemical interconversions that sustain life. What is a Enzyme ？

Etymology and history In 1833, French chemist Anselme Payen discovered the first enzyme, diastase. In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Greek ενζυμον, “in leaven” (in yeast). In 1897, Eduard Buchner discovered cell-free fermentation "zymase". (1907 Nobel Prize in Chemistry) In 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it (1946 Nobel Prize in Chemistry). In 1965, the first X-ray 3D structure of enzyme was done for lysozyme by David Chilton Phillips’ group. In 1989, Thomas R. Cech and Sidney Altman won the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA.“ in 1980s.

Substrates Enzyme Product Enzymatic reactions 1.In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. 2.Enzymes are used commercially, for example, in the synthesis of antibiotics, in biological washing powders break down protein or fat stains on clothes, and in food industry to break down proteins.

Enzymes catalyze the conversion of substrates into products What is a substrate? –A substrate is the compound that is converted into the product in an enzyme catalyzed reaction. –For the reaction catalyzed by aldolase, fructose 1,6-phosphate is the substrate.

What are enzymes made from? Protein 1.All enzymes are proteins except some RNAs (Ribozymes) and DNAs (deoxyribozymes) 2.not all proteins are enzymes Nucleic acid Enzyme

Ribozymes  It was assumed that all enzymes are proteins until 1982 when Thomas Cech and Sydney Altman discovered catalytic RNAs (Nobel, 1989 in Chemistry);  The RNA world hypothesis, if true, has important implications for the definition of life, places RNA at center-stage when life originated.

What do enzymes do? Enzymes are biological catalysts that accelerate the rates of chemical reactions. Snail without enzyme catalyst Snail with enzyme catalyst

Enzyme catalyzed reactions are much faster than non-catalytic reactions. Time [Product] Enzyme catalyzed reaction Non-catalytic reaction 0 0 Reaction Rate =  [Product]  (time)

General Properties of Enzymes 1. Higher reaction rates : carbonic anhydrase CO 2 +H 2 O  H 2 CO 3 nonenzymatic rate constant = 1.3 x s -1 enzymatic rate constant = 1 x 10 6 s -1 (x 7.7 x 10 6 ) Staphylococcal nuclease nonenzymatic rate constant = 1.7 x s -1 enzymatic rate constant = 95 s -1 (x 5.6 x )

Enzymes do more than just increase the rate of a chemical reaction  Control when and where reactions occur  Regulate the rate of a reaction (“controlled combustion”)  Prevent unwanted side reactions  Optimize reaction for specific conditions (e.g. pH, temperature)

Some definitions of the enzyme  Monomeric enzyme: only one polypeptide chain in which the active site resides.  Oligomeric enzyme ： More than one polypeptide / subunits 。  Multienzyme system ： System of two or more enzymes functioning sequentially to catalyze the reactions  Multifunctional enzyme or tandem enzyme ： Enzyme with more than one catalytic activity  Isozymes or isoenzymes or multiple forms of enzymes: enzymes that differ in amino acid sequence but catalyze the same chemical reaction.

Isozymes: An automotive analogy 1.Physically distinct forms of the same enzyme. 2.Differ in amino acid sequences or posttranslational modifications 3.Different tissues or subcellular organelles

Example: Lactate Dehydrogenase is composed of four monomers  Homo or hetero tetramers composed of muscle (M) and heart (H) protein subunits encoded by the LDHA and LDHB genes, respectively:  LDHx—A third isoform, coded by LDHC or LDHX gene, is expressed only in the testis  LDH-1 (4H)—in the heart and in RBC (red blood cells)  LDH-2 (3H1M)—in the reticuloendothelial system  LDH-3 (2H2M)—in the lungs  LDH-4 (1H3M)—in the kidneys, placenta, and pancreas  LDH-5 (4M)—in the liver and striated muscle HH HH HH HM HH MM H MM M MM MM LDH 1 (H 4 ) LDH 2 (H 3 M) LDH 3 (H 2 M 2 ) LDH 4 (HM 3 ) LDH 5 (M 4 )

Components of the enzyme A poenzyme: the protein part of an enzyme Cofactor Inorganic: Metal ions Organic Holoenzyme Simple enzyme Conjugated enzyme Prosthetic group: emphasizes the nature of the binding of a cofactor to a protein (tight or covalent) Coenzymes: additional substance required for enzyme activity

Active site of Enzyme Definition: The small 3D groove or pocket of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site displays highly specific substrate binding The active site is responsible for whether there is ordered or random binding of substrates and release of products

The active site of the enzyme Active site of Chymotrypsin (a digestive enzyme component of pancreatic juice acting in the duodenum where it performs proteolysis, the breakdown of proteins and polypeptides.)

Important sites of Enzyme Active site residues: residues that directly participate in the catalytic reaction mechanism in active site of enzyme. Essential groups: Side chain groups required for the catalytic ability of enzymes  Inside active site: for reaction binding group catalytic group  Outside active site: for conformation

Substrate Outside the active site Binding group Catalytic groups Active site

Specificity of Enzyme  Absolute specificity: specific substrate  Relative specificity ： a class of bond or group  Stereospecificity ： specific stereoisomer

Enzyme active sites can distinguish between stereoisomers Stereoisomers are non-superimposable mirror images of each other

Enzymes contain binding sites that recognize the substrates of the reaction

Enzyme Nomenclature and Classes  Oxidoreductases(EC Class 1) Transfer electrons (RedOx reactions)  Transferases(EC Class 2) Transfer functional groups between molecules  Hydrolases(EC Class 3) Break bonds by adding H 2 O  Lyases(EC Class 4) Elimination reactions to form double bonds  Isomerases(EC Class 5) Intramolecular rearangements  Synthetase or Ligases(EC Class 6) Join molecules with new bonds

Oxidoreductases catalyze the transfer of hydrogen atoms and electrons  Example - Lactate Dehydrogenase O O - C C CH 3 O + NADH +H + O O - CH C CH 3 HO + pyruvateL-lactate lactate dehydrogenase

Transferases catalyze the transfer of functional groups from donors to acceptors  Example - Alanine aminotransferase O O - C C CH 3 O pyruvate O O - CH C CH 2 H 2 N CH 2 O O - C glutamate O O - CH C CH 3 H 2 N L-alanine O O - C C CH 2 CH 2 O O - C O  -ketoglutarate ++ alanine aminotransferase

Hydrolases catalyze the cleavage of bonds by the addition of water (hydrolysis)  Example - Trypsin N H C NH 2 (CH 2 ) 4 H O C O HH C N H N H C CH 3 H O C O H C N H CC CH(CH 3 ) 2 N H C NH 2 (CH 2 ) 4 H O C O HH C N H O - C N H C CH 3 H O C O CH(CH 3 ) 2 H C H 3 N + C + H 2 O trypsin + Gly-Lys-Val-Ala Val-Ala Gly-Lys

Lyases catalyze the cleavage of C-C, C-O, or C-N bonds (addition of groups to double bonds or formation of double bonds by removal of groups)  Example - ATP-citrate lyase O O - C C CH 2 O O O - C oxaloacetate O O - CH 2 C CH 2 O O - C C O O - C HO citrate O CH 3 C O - acetate ATP-citrate lyase + ATP ADP + P i Coenzyme A

Isomerases catalyze the transfer of functional groups within the same molecule  Example - Phosphoglucose isomerase

Ligases use ATP to catalyze the formation of new covalent bonds  Example - DNA ligase

Substrate + EnzymeES complex Enzyme Substrate + ES complex Lock and Key - Emil Fischer (1890) Induced Fit - Daniel E. Koshland Jr. (1958)

An Example: Induced conformational change in hexokinase Catalyzes phosphorylation of glucose to glucose 6-phosphate during glycolysis such a large change in a protein’s conformation is not unusual BUT: not all enzymes undergo such large changes in conformation

Advantage of the induced fit mechanism  The active site can be open to allow substrates to bind, then close over the substrates to provide optimum transition state stabilization Disadvantage of the induced fit mechanism  Energy that would otherwise be used to help stabilize the transition state of the reaction must be used to induce the conformational change in the enzyme.

Greater capacity for regulation of Enzymes Enzymes can respond to allosteric compounds that alter their kinetic properties. Enzymes can also be regulated by covalent modification : e.g. phosphorylation can inactivate or activate an enzyme. Through increased transcription of the gene encoding a particular enzyme, the level of mRNA for the enzyme can increased. This can increase levels of this enzyme.

Important things to remember about enzymes (just like other catalysts) 1. Enzymes are not consumed or altered by the reaction they catalyze. Just as a construction worker can take a pile of lumber and build a home without being physically changed by the process, an enzyme takes substrates and converts them into products without being physically changed or consumed.

This is an important point. An enzyme does not determine which direction the reaction goes, it only increases the rate at which the reaction approaches equilibrium. 2. Enzymes catalyze both the forward and the reverse reaction.

3. Enzymes do not alter the equilibrium (or equilibrium constant) between substrates and products. At equilibrium, the ratio of substrates to products is the same regardless of whether an enzyme catalyst is present. Although the final equilibrium ratio of reactants to products is not altered by an enzyme, the rate at equilibrium is achieved is increased.

 G < 0 for the conversion of diamond into graphite Thermodynamics vs. Kinetics Thermodynamics tells us whether a reaction can occur. Kinetics tells us whether the reaction will occur in our lifetimes.

Section two: Thermodynamics and Kinetics of Enzyme-Catalyzed Reaction Yes/No? Now/Later?

What is the transition state? The transition state is the most unstable species on the reaction coordinate (i.e. the species with the highest energy).. Substrates Products Transition State  G rxn Free Energy ( GG ) Reaction Coordinate

Transition State Theory  Reactants are required to reach a high-energy (unstable) state referred to as the transition state. Residence may last only to s  Only a limited number of molecules will possess sufficient energy to reach this transition state.  Increasing temperature or other conditions may facilitate reactants reaching the transition state

The transition state is not an intermediate species The transition state cannot be trapped or isolated. Intermediates can be trapped or isolated... Products Transition State Free Energy ( GG ) Reaction Coordinate Substrates Intermediate

The Transition State  Understand the difference between  G and  G ‡ (Gibbs energy)  The overall free energy change for a reaction (∆G) is related to the equilibrium ratio of [S] and [P]  The free energy of activation for a reaction (  G ‡ ) is related to the reaction rate  It is extremely important to appreciate this distinction!

Free energy tells us nothing about the rate at which the reaction occurs The height of the transition state relative to the reactants determines how rapidly substrates are converted to product.. Substrates Products Transition State  G rxn Free Energy ( GG ) Reaction Coordinate With Enzyme G‡G‡

General Properties of Enzymes Enzymes bind substrates to their active site and stabilize the transition state of the reaction.

Enzyme Kinetics The rate of unimolecular reaction is proportional to the concentration of the reactant. Thus rate is linearly dependent on [A]. But if this reaction is catalyzed by an enzyme, the rate shows saturation behavior. Why? v [A] v

[S] V V max [S] V [S] V Early stage Middle stage Saturated stage

Leonor Michaelis ( ) Maud Menten ( )

The Michaelis–Menten Equation You’d better know how this is derived This is the complete chemical formula for an enzyme- catalyzed (E) reaction of substrate, S and product, P; Michaelis–Menten equation describes the relationship between reaction rate and substrate concentration. Assumptions for Mechaelis-Menten: –Initial velocity assumption –Rate law –State steady assumption k1k1 K -1 k2k2 K -2

Initial Velocity Assumption In the beginning of the reaction, there is very little product, or [P] is small. So the amount of [ES] contributed by E+P is negligible. Thus, the MM equation concerns the reaction rate that is measured during early reaction period. In which case, the enzyme catalyzed reaction can be simplified to: k1k1 K -1 k2k2 k1k1 k2k2 K -2

Rate Law and Steady state in Enzyme Catalyzed Reactions Rate law still applies in enzyme catalyzed reactions. The forward velocity, or rate, v f is, The reverse velocity or rate, or the rate of disappearance v d is, At steady state, there is no accumulation of [ES], thus: k1k1 K -1 k2k2

Derivation of Michaelis-Menten Equation We need one more condition, that is, the total enzyme concentration, [E t ] is the sum of that of enzyme- substrate complex, [ES], and that of free enzyme, [E]: At steady state, the forward rate should equal to the reverse rate: Rate of production formation (rate law), v = k 2 [ES]. So:

Understanding K m K m is a constant derived from rate constants K m is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S, because at equilibrium, Reversible reaction, dissociation constant is So small K m means tight substrate binding; high K m means weak substrate binding. K m equals to the substrate concentration at which v=v max /2 k1k1 k -1

Notes on the MM Equations The rate of production formation can usually be measured experimentally by monitoring the progress curve of production formation. The maximum rate can be reached at saturating substrate concentration, or when [S] So MM equation can be re-written as: Enzyme-catalyzed rate is saturated

Understanding V max The theoretical maximal velocity  V max is a constant  V max is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality  To reach V max would require that ALL enzyme molecules are tightly bound with substrate  V max is asymptotically approached as substrate is increased

The dual nature of the Michaelis-Menten equation  The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!

The turnover number A measure of catalytic activity  k cat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate.  If the M-M model fits, k 2 = k cat = V max /E t  Values of k cat range from less than 1/sec to many millions per sec

The catalytic efficiency Name for k cat /K m  An estimate of "how perfect" the enzyme is  k cat /K m is an apparent second-order rate constant  It measures how the enzyme performs when S is low  The upper limit for k cat /K m is the diffusion limit - the rate at which E and S diffuse together

1 V = 1 V MAX + KMKM 1 [S] y = m x + b x intercept = - 1 / K M 0 1 / [S] 1 / V y intercept = 1 / V MAX Determining the VMAX and KM : Lineweaver-Burk plot Take reciprocal for both sides Denominators ！

Enzyme catalyzed reactions are optimized for specific values of temperature and pH pH Reaction Rate Enzyme catalyzed reaction Non-catalytic reaction Temperature ( o C) Reaction Rate Enzyme catalyzed reaction non-catalytic reaction

Enzyme Inhibition Reversible inhibitors associate with enzymes through non-covalent interactions. Reversible inhibitors include three kinds: Competitive inhibitors; Non-competitive inhibitors; Un-competitive inhibitors Irreversible inhibitors associate with enzymes through covalent interactions. Thus the consequences of irreversible inhibitors is to decrease in the concentration of active enzymes.

Competitve Inhibitors

 Often “resemble” substrates or cofactors  Bind to enzyme through similar complementary interactions  Example: Dihydrofolate reductase

Competitive Inhibitors v [S][S] v max KmKm 1/[S] 1/v 1/v max -1/K m Slope=K m /v max k1k1 K -1 k2k2 KIKI K m increases v max unchanged +inhibitor K m (1+[I]/K I ) -1/(K m (1+[I]/K I )) Slope= K m (1+[I]/K I )/v max

Non-Competitve Inhibitors

Noncompetitive Inhibitors k1k1 k -1 k2k2 KIKI K m unchanged v max decreases K I’ +inhibitor 1/[S] 1/v 1/v max -1/K m Slope=K m /v max (1+[I]/K I )/V max Slope= K m (1+[I]/K I )/v max v [S][S] v max KmKm KmKm V max /(1+[I]/K I )

Uncompetitive Inhibitors k1k1 k -1 k2k2 K m decreases v max decreases Slope unchanged +inhibitor K I’ 1/[S] 1/v 1/v max -1/K m Slope=K m /v max (1+ K I /[I])/V max Slope= K m /v max v [S][S] v max KmKm K m /(1+ K I /[I]) V max /(1+K I /[I]) - (1+ K I /[I])/K m

Summary of Classes of reversible Inhibitors Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition. Mixed inhibition-when the dissociation constants of (I) to E and ES are different. The inhibition is mixed.

Irreversible Inhibitor  Combines with or destroys an essential functional group on the enzyme (e.g. forms covalent bonds)  Inhibit enzymes irreversibly  3 different types: –Group Specific Reagent: - inhibitor does not resemble substrate –Substrate Analogue: - inhibitor resembles substrate –Suicide Inhibitors: - inhibitor resembles substrate, turns "dangerous" after processed by enzyme

Lewisite Enzyme lost activity desulfhydrase Enzyme lost activity BAL desulfhydrase Toxication and detoxication Irreversible does not mean No cure! ——Irreversible does not mean No cure!

Group Specific Reagent  Does not resemble substrate  irreversibly inactivates enzyme by modifying an essential R group  e.g. DIPF (potent nerve gas) blocks acetylcholinesterase, which is essential for proper neural transmission active site serine

Substrate Analogue  Binds at enzyme active site  Then irreversibly modifies (or binds to) to the active site

Penicillin is a suicide inhibitor  Resemble substrate  Binds at enzyme active site (not an "inhibitor" yet)  Processed by enzyme via normal catalytic mechanism to a chemically active intermediates that inactivates the enzyme irreversibly  Good candidate for drug due to minimal side effect Penicillin was discovered by the Scottish doctor Alexander Fleming in 1928, antibiotics for bacterial by inhibiting cell wall formation. (the Nobel prize in Medicine).