Reginald H. Garrett Charles M. Grisham Chapter 14 Mechanisms of Enzyme Action

Chapter 14 “No single thing abides but all things flow. Fragment to fragment clings and thus they grow. Until we know them by name. Then by degrees they change and are no more the things we know.” Lucretius ca. 94 B.C. – 50 B.C. Like the workings of machines, the details of enzyme mechanisms are once complex and simple.

Essential Questions What are the universal chemical principles that influence the mechanisms of enzymes and allow us to understand their enormous catalytic power?

Outline What are the magnitudes of enzyme-induced rate accelerations? What role does transition-state stabilization play in enzyme catalysis? How does destabilization of ES affect enzyme catalysis? How tightly do transition-state analogs bind to the active site? What are the mechanisms of catalysis? What can be learned from typical enzyme mechanisms?

14.1 What Are the Magnitudes of Enzyme- Induced Rate Accelerations? Enzymes are powerful catalysts The large rate accelerations of enzymes (107 to 1015) correspond to large changes in the free energy of activation for the reaction All reactions pass through a transition state on the reaction pathway The active sites of enzymes bind the transition state of the reaction more tightly than the substrate By doing so, enzymes stabilize the transition state and lower the activation energy of the reaction

14.1 What Are the Magnitudes of Enzyme- Induced Rate Accelerations?

14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis? The catalytic role of an enzyme is to reduce the energy barrier between substrate S and transition state Rate acceleration by an enzyme means that the energy barrier between ES and EX ‡ must be smaller than the barrier between S and X ‡ This means that the enzyme must stabilize the EX ‡ transition state more than it stabilizes ES See Eq. 14.3

14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis? Figure 14.1 Enzymes catalyze reactions by lowering the activation energy. Here the free energy of activation for (a) the uncatalyzed reaction is larger than that of the enzyme-catalyzed reaction.

14.2 What Role Does Transition-State Stabilization Play in Enzyme Catalysis? Competing effects determine the position of ES on the energy scale Try to mentally decompose the binding effects at the active site into favorable and unfavorable The binding of S to E must be favorable But not too favorable! K m cannot be "too tight" - goal is to make the energy barrier between ES and EX ‡ small

14.3 How Does Destabilization of ES Affect Enzyme Catalysis? Raising the energy of ES raises the rate For a given energy of EX ‡, raising the energy of ES will increase the catalyzed rate This is accomplished by a) loss of entropy due to formation of ES b) destabilization of ES by strain distortion desolvation

14.3 How Does Destabilization of ES Affect Enzyme Catalysis? Figure 14.2 The intrinsic binding energy of ES is compensated by entropy loss due to binding of E and S and by destabilization due to strain and distortion.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis? Figure 14.3 (a) Catalysis does not occur if ES and X ‡ are equally stabilized. (b) Catalysis will occur if X ‡ is stabilized more than ES.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis? Figure 14.4 (a) Formation of the ES complex results in entropy loss. The ES complex is a more highly ordered, low-entropy state for the substrate.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis? Figure 14.4 (b) Substrates typically lose waters of hydration in the formation in the formation of the ES complex. Desolvation raises the energy of the ES complex, making it more reactive.

14.3 How Does Destabilization of ES Affect Enzyme Catalysis? Figure 14.4 (c) Electrostatic destabilization of a substrate may arise from juxtaposition of like charges in the active site. If charge repulsion is relieved in the reaction, electrostatic destabilization can result in a rate increase.

14.4 How Tightly Do Transition-State Analogs Bind to the Active Site? Very tight binding to the active site The affinity of the enzyme for the transition state may be to M! Can we see anything like that with stable molecules? Transition state analogs (TSAs) are stable molecules that are chemically and structurally similar to the transition state Proline racemase was the first case See Figure 14.6 for some recent cases

14.4 How Tightly Do Transition-State Analogs Bind to the Active Site? Figure 14.5 The proline racemase reaction. Pyrrole-2- carboxylate and Δ-1-pyrroline-2-carboxylate mimic the planar transition state of the reaction.

14.4 How Tightly Do Transition-State Analogs Bind to the Active Site? Figure 14.6 (a) Phosphoglycolohydroxamate is an analog of the enediolate transition state of the yeast aldolase reaction.

14.4 How Tightly Do Transition-State Analogs Bind to the Active Site? (b) Purine riboside inhibits adenosine deaminase. The hydrated form is an analog of the transition state of the reaction.

Transition-State Analogs Make Our World Better Enzymes are often targets for drugs and other beneficial agents Transition state analogs often make ideal enzyme inhibitors Enalapril and Aliskiren lower blood pressure Statins lower serum cholesterol Protease inhibitors are AIDS drugs Juvenile hormone esterase is a pesticide target Tamiflu is a viral neuraminidase inhibitor

Transition-State Analogs Make Our World Better High blood pressure is a significant risk for cardiovascular disease. Transition-state analog drugs reduce the risk of heart attacks.

Transition-State Analogs Make Our World Better Blood pressure is partly regulated by aldosterone, a steroid made and released in blood vessels by angiotensin II, a peptide produced from angiotensinogen in two proteolytic steps by renin and ACE. Enalapril is an ACE inhibitor. Aliskiren is a renin inhibitor. Both are TSAs.

Transition-State Analogs Make Our World Better Statins such as Lipitor are powerful cholesterol-lowering drugs, because they are transition-state analog inhibitors of HMG-CoA reductase, a key enzyme in the biosynthetic pathway for cholesterol.

Transition-State Analogs Make Our World Better Statins such as Lipitor are powerful cholesterol-lowering drugs, because they are transition-state analog inhibitors of HMG-CoA reductase, a key enzyme in the biosynthetic pathway for cholesterol.

Transition-State Analogs Make Our World Better Invirase (saquinavir) by Roche and similar “protease inhibitor” drugs are transition-state analogs for the HIV-1 protease.

Transition-State Analogs Make Our World Better Insects have significant effects on human health. Malaria, West Nile virus, and viral encephalitis are carried by mosquitoes (left). Lyme disease and Rocky Mountain spotted fever are carried by ticks (right).

Transition-State Analogs Make Our World Better One strategy for controlling insect populations is to alter the actions of juvenile hormone, a terpene-based substance that regulates insect life cycle processes. Levels of juvenile hormone are controlled by juvenile hormone esterase (JHE), and inhibition of JHE is toxic to insects. OTEP (figure) is a potent transition state analog inhibitor of JHE.

The 1918 flu pandemic killed more than 20 million people worldwide.

Tamiflu is a Viral Neuraminidase Inhibitor Influenza is a serious illness that affects 5% to 15% of the earth’s population each year and results in up to 500,000 deaths annually. Neuraminidase is a major glycoprotein on the influenza virus membrane envelope that is essential for viral replication and infectivity. Tamiflu is a neuraminidase inhibitor and antiviral agent based on the transition state of the neuraminidase reaction.

How many other drug targets might there be? The human genome contains approximately 20,000 genes How many might be targets for drug therapy? More than 3000 experimental drugs are presently under study and testing These and many future drugs will be designed as transition-state analog inhibitors See the DrugBank:

How to read and write mechanisms The custom of writing chemical reaction mechanisms with electron dots and curved arrows began with Gilbert Newton Lewis and Sir Robert Robinson Learning to read and write mechanisms should begin with a review of Lewis dot structures And with a review of the concepts of valence electrons and formal charge Formal charge = group number – nonbonding electrons – (1/2 shared electrons) Electronegativity is also important: F > O > N > C > H

How to read and write mechanisms In written mechanisms, a curved arrow shows the movement of an electron pair And thus the movement of a pair of electrons from a filled orbital to an empty one A full arrowhead represents an electron pair A half arrowhead represents a single electron For a bond-breaking event, the arrow begins in the middle of the bond

How to read and write mechanisms For a bond-breaking event, the arrow begins in the middle of the bond, and the arrow points to the atom that will accept the electrons.

How to read and write mechanisms For a bond-making event, the arrow begins at the source of the electrons (for example, a nonbonded pair), and the arrowhead points to the atom where the new bond will be formed.

How to read and write mechanisms It has been estimated that 75% of the steps in enzyme reaction mechanisms are proton (H + ) transfers. If the proton is donated or accepted by a group on the enzyme, it is often convenient (and traditional) to represent the group as “B”, for “base”, even if B is protonated and behaving as an acid:

How to read and write mechanisms It is important to appreciate that a proton transfer can change a nucleophile into an electrophile, and vice versa. Thus, it is necessary to consider: The protonation states of substrate and active-site residues How pK a values can change in the environment of the active site For example, an active-site histidine, which might normally be protonated, can be deprotonated by another group and then act as a base, accepting a proton from the substrate

How to read and write mechanisms An active-site histidine, which might normally be protonated, can be deprotonated by another group and then act as a base, accepting a proton from the substrate.

How to read and write mechanisms Water can often act as an acid or base at the active site through proton transfer with an assisting active-site residue: This type of chemistry is the basis for general acid-base catalysis (discussed on pages ).

14.5 What Are the Mechanisms of Catalysis? Enzymes facilitate formation of near-attack complexes Protein motions are essential to enzyme eatalysis Covalent catalysis General acid-base catalysis Low-barrier hydrogen bonds Metal ion catalysis

Enzymes facilitate formation of near-attack complexes X-ray crystal structure studies and computer modeling have shown that the reacting atoms and catalytic groups are precisely positioned for their roles Such preorganization selects substrate conformations in which the reacting atoms are in van der Waals contact and at an angle resembling the bond to be formed in the transition state Thomas Bruice has termed such arrangements near- attack conformations (NACs) NACs are precursors to reaction transition states

Enzymes facilitate formation of near-attack complexes Thomas Bruice has proposed that near-attack conformations are precursors to transition states In the absence of an enzyme, potential reactant molecules adopt a NAC only about % of the time On the other hand, NACs have been shown to form in enzyme active sites from 1% to 70% of the time

Enzymes facilitate formation of near- attack complexes Figure 14.7 NACs are characterized as having reacting atoms within 3.2 Å and an approach angle of ±15° of the bonding angle in the transition state.

Enzymes facilitate formation of near-attack complexes Figure 14.7 In an enzyme active site, the NAC forms more readily than in the uncatalyzed reaction. The energy separation between the NAC and the transition state is approximately the same in the presence and absence of the enzyme.

Figure 14.8 The active site of liver alcohol dehydrogenase – a near-attack complex.

Protein Motions Are Essential to Enzyme Catalysis Proteins are constantly moving – bonds vibrate, side chains bend and rotate, backbone loops wiggle and sway, and whole domains move as a unit Enzymes depend on such motions to provoke and direct catalytic events Protein motions support catalysis in several ways. Active site conformation changes can: Assist substrate binding Bring catalytic groups into position Induce formation of NACs Assist in bond making and bond breaking Facilitate conversion of substrate to product

Protein Motions Are Essential to Enzyme Catalysis Figure 14.9 Human cyclophilin A is a prolyl isomerase, which catalyzes the interconversion between trans and cis conformations of proline in peptides.

Protein Motions Are Essential to Enzyme Catalysis Figure 14.9 The active site of cyclophilin with a bound peptide containing proline in cis and trans conformations. Motion by active site residues promote catalysis in cyclophilin.

Protein Motions Are Essential to Enzyme Catalysis Figure Catalysis in enyzme active sites depends on motion of active-site residues. Several active-site residues undergo greater motion during catalysis than residues elsewhere in the protein.

Covalent Catalysis Some enzymes derive much of their rate acceleration from formation of covalent bonds between enzyme and substrate The side chains of amino acids in proteins offer a variety of nucleophilic centers for catalysis These groups readily attack electrophilic centers of substrates, forming covalent enzyme-substrate complexes The covalent intermediate can be attacked in a second step by water or by a second substrate, forming the desired product

Covalent Catalysis Figure Examples of covalent enzyme-substrate intermediates.

Covalent Catalysis Figure Examples of covalent bond formation between enzyme and substrate. A nucleophilic center X: on an enzyme attacks a phosphorus atom to form a phosphoryl enzyme intermediate.

Covalent Catalysis Figure Examples of covalent bond formation between enzyme and substrate. A nucleophilic center X: on an enzyme attacks a carbonyl C to form an acyl enyzme intermediate.

Covalent Catalysis Figure Examples of covalent bond formation between enzyme and substrate. A nucleophilic center X: attacks the anomeric carbon of a glycoside, forming a glucosyl enzyme intermediate.

Covalent Catalysis

General Acid-base Catalysis Catalysis in which a proton is transferred in the transition state "Specific" acid-base catalysis involves H + or OH - that diffuses into the catalytic center "General" acid-base catalysis involves acids and bases other than H + and OH - These other acids and bases facilitate transfer of H + in the transition state See Figure 14.12

General Acid-base Catalysis Figure Catalysis of p-nitrophenylacetate hydrolysis can occur either by specific acid hydrolysis or by general base catalysis.

Low-Barrier Hydrogen Bonds (LBHBs) The typical H-bond strength is kJ/mol, and the O-O separation is typically 0.28 nm As distance between heteroatoms becomes smaller (<0.25 nm), H bonds become stronger Stabilization energies can approach 60 kJ/mol in solution pK a values of the two electronegative atoms must be similar Energy released in forming an LBHB can assist catalysis

Low-Barrier Hydrogen Bonds (LBHBs) Figure Energy diagrams for conventional H bonds (a), and low-barrier hydrogen bonds (b and c).s In (c), the O-O distance is 0.23 to 0.24 nm, and bond order for each O-H interaction is 0.5.

Metal Ion Catalysis Figure Thermolysin is an endoprotease with a catalytic Zn 2+ ion in the active site. The Zn 2+ ion stabilizes the buildup of negative charge on the peptide carbonyl oxygen, as a glutamate residue deprotonates water, promoting hydroxide attack on the carbonyl carbon.

How Do Active-Site Residues Interact to Support Catalysis? About half of the amino acids engage directly in catalytic effects in enzyme active sites Other residues may function in secondary roles in the active site: Raising or lowering catalytic residue pK a values Orientation of catalytic residues Charge stabilization Proton transfers via hydrogen tunneling

How Do Active-Site Residues Interact to Support Catalysis? The active site of aromatic amine dehydrogenase, showing the relationship of Asp 128, Thr 172, and Cys 171. Coupling of local motions of these residues to vibrational states involved in proton transfer contributes to catalysis.

14.5 What Can Be Learned From Typical Enzyme Mechanisms? First Example: the serine proteases Enzyme and substrate become linked in a covalent bond at one or more points in the reaction pathway The formation of the covalent bond provides chemistry that speeds the reaction Serine proteases also employ general acid-base catalysis

The Serine Proteases Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA All involve a serine in catalysis - thus the name Ser is part of a "catalytic triad" of Ser, His, Asp Serine proteases are homologous, but locations of the three crucial residues differ somewhat Enzymologists agree, however, to number them always as His 57, Asp 102, Ser 195 Burst kinetics yield a hint of how they work

The Serine Proteases Figure The amino acid sequences of chymotrypsinogen, trypsin, and elastase.

The Catalytic Triad of the Serine Proteases Figure Structure of chymotrypsin (white) in a complex with eglin C (blue ribbon structure), a target substrate. His 57 (red) is flanked by Asp 102 (gold) and Ser 195 (green). The catalytic site is filled by a peptide segment of eglin. Note how close Ser 195 is to the peptide that would be cleaved in the reaction.

The Catalytic Triad of the Serine Proteases Figure The catalytic triad at the active site of chymotrypsin (and the other serine proteases.

Serine Protease Binding Pockets are Adapted to Particular Substrates Figure The substrate-binding pockets of trypsin, chymotrypsin, and elastase. Asp 189 (aqua) coordinates Arg and Lys residues of substrates in the trypsin pocket. Val 216 (purple) and Thr 226 (green) make the elastase pocket shallow and able to accommodate only small, nonbulky residues. The chymotrypsin pocket is hydrophobic.

Serine Proteases Cleave Simple Organic Esters, such as p-Nitrophenylacetate Figure Chymotrypsin cleaves simple esters, in addition to peptide bonds. P-Nitrophenylacetate has been used in studies of the chymotrypsin mechanism.

Serine Proteases Display Burst Kinetics Figure Burst kinetics in the chymotrypsin reaction.

Serine Protease Mechanism A mixture of covalent and general acid-base catalysis Asp 102 functions only to orient His 57 His 57 acts as a general acid and base Ser 195 forms a covalent bond with peptide to be cleaved Covalent bond formation turns a trigonal C into a tetrahedral C The tetrahedral oxyanion intermediate is stabilized by N-Hs of Gly 193 and Ser 195

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: binding of a model substrate.

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: the formation of the covalent ES complex involves general base catalysis by His 57

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: His 57 stabilized by a LBHB.

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: collapse of the tetrahedral intermediate releases the first product.

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: The amino product departs, making room for an entering water molecule.

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: Nucleophilic attack by water is facilitated by His 57, acting as a general base.

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: Collapse of the tetrahedral intermediate cleaves the covalent intermediate, releasing the second product.

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: Carboxyl product release completes the serine protease mechanism.

The Serine Protease Mechanism in Detail Figure The chymotrypsin mechanism: At the completion of the reaction, the side chains of the catalytic triad are restored to their original states.

Transition-State Stabilization in the Serine Proteases The chymotrypsin mechanism involves two tetrahedral oxyanion intermediates These intermediates are stabilized by a pair of amide groups that is termed the “oxyanion hole” The amide N-H groups of Ser 195 and Gly 193 provide primary stabilization of the tetrahedral oxyanion

The “oxyanion hole” The oxyanion hole of chymotrypsin stabilizes the tetrahedral oxyanion intermediate seen in the mechanism of Figure

Aspartic proteases play many roles in humans

The Aspartic Proteases Pepsin, chymosin, cathepsin D, renin and HIV-1 protease All involve two Asp residues at the active site These two Asp residues work together as general acid-base catalysts Most aspartic proteases have a tertiary structure consisting of two lobes (N-terminal and C-terminal) with approximate two-fold symmetry HIV-1 protease is a homodimer

The Aspartic Proteases Figure Structures of (a) HIV-1 protease, a dimer, and (b) pepsin, a monomer. Pepsin’s N- terminal half is shown in red; the C-terminal half is shown in blue. Most aspartic proteases exhibit a two-lobed structure. Each lobe contributes one catalytic aspartate to the active site. HIV-1 protease is a homodimeric enzyme, with each subunit contributing a catalytic Asp residue.

The Aspartic Proteases Figure pH- rate profile for pepsin.

The Aspartic Proteases Figure pH-rate profile of HIV-1 protease.

Aspartic Protease Mechanism Aspartic proteases show one relatively low pK a, and one relatively high pK a This was once thought to represent pK a values of the two aspartate residues, but this is no longer believed to be the case Instead, molecular dynamics simulations show that aspartic proteases employ low-barrier hydrogen bonds (LBHBs) in their mechanism The predominant catalytic factor in aspartic proteases is general acid-base catalysis

A Mechanism for the Aspartic Proteases Figure Mechanism for the aspartic proteases. LBHBs play a role in states E, ES, ET’, EQ’, and EP’Q.

Aspartic Proteases May Employ Hydrogen Tunneling for Rate Acceleration Figure Energy level diagram for the aspartic protease reaction, showing hydrogen tunneling.

HIV-1 Protease A novel aspartic protease HIV-1 protease cleaves the polyprotein products of the HIV genome This is a remarkable imitation of mammalian aspartic proteases HIV-1 protease is a homodimer - more genetically economical for the virus Active site is two-fold symmetric

Proteolytic cleavage pattern for the HIV genome Figure HIV mRNA provides the genetic information for synthesis of a polyprotein. Cleavage yields the active products.

Protease Inhibitors Block the Active Site of HIV-1 Protease Figure HIV-1 protease complexed with the inhibitor Crixivan (red) made by Merck. The “flaps” that cover the active site are green; the catalytic active site Asp residues are violet.

Protease Inhibitors Give Life to AIDS Patients Protease inhibitors as AIDS drugs If the HIV-1 protease can be selectively inhibited, then new HIV particles cannot form Several novel protease inhibitors are currently marketed as AIDS drugs Many such inhibitors work in a culture dish However, a successful drug must be able to kill the virus in a human subject without blocking other essential proteases in the body

Protease Inhibitors Give Life to AIDS Patients Protease inhibitor drugs used by AIDS Patients

Chorismate Mutase: A Model for Understanding Catalytic Power and Efficiency Direct comparison of enzyme-catalyzed reactions and their uncatalyzed counterparts is difficult Chorismate mutase has become a model for making this comparison, thanks to the efforts of a large number of enzyme mechanism researchers Chorismate mutase acts in the biosynthesis of phenylalanine and tyrosine in microorganisms and plants It involves a single substrate and catalyzes a concerted intramolecular rearrangement of chorismate to prephenate One C-O bond is broken and one C-C bond is formed

Chorismate Mutase: A Model for Understanding Catalytic Power and Efficiency Figure The chorismate mutase reaction converts chorismate to prephenate in an intramolecular rearrangement.

Chorismate Mutase: A Model for Understanding Catalytic Power and Efficiency Figure A classic Claisen rearrangement. Conversion of allyl phenyl ether to 2-allyl alcohol proceeds through a cyclohexadienone intermediate, which then undergoes a keto- enol tautomerization.

The chorismate mutase reaction (and its uncatalyzed counterpart) occur via chair states Figure The critical H atoms are distinguished in this figure by blue and green colors.

A transition-state analog for the chair mechanism of chorismate mutase Jeremy Knowles has shown that both the chorismate mutase and its uncatalyzed solution counterpart proceed via a chair mechanism. A transition state analog of this state has been characterized.

The structure of E. coli chorismate mutase Figure (a) the chorismate mutase homodimer (b) The active site, showing the bound transition-state analog.

Transition state stabilization by electrostatic and hydrogen-bonding interactions Figure Twelve electrostatic and hydrogen-bonding interactions stabilize the transition-state analog.

The Chorismate Mutase Mechanism Figure The carboxyvinyl group folds up and over the chorismate ring and the reaction proceeds via an internal rearrangement.

The Chorismate Mutase Active Site Favors a Near-Attack Conformation Figure Chorismate boudn to the active site of chorismate mutase in a structure that resembles a near-attack complex. Arrows indicate hydrophobic interactions and red dotted lines indicate electrostatic interactions.

Formation of a NAC is facile in the chorismate mutase active site Figure Chorismate mutase facilitates NAC formation. The energy required to move from the NAC to the transition state is essentially equivalent in the catalyzed and uncatalyzed reactions.

A High-Energy Intermediate in the Phosphoglucomutase Reaction Is this a demonstration of a transition state in a crystal structure?