Mechanisms of Enzyme Action

What You Need to Know• Understand the importance of and need for enzymes in

biological reactions.• Understand how an enzyme’s effect on the transition state and

destabilization of the enzyme-substrate complex can affect reaction rates.

• What role does transition-state stabilization play in enzyme catalysis?

• Why is protein motion important in enzyme catalysis?• Understand importance of near attack conformations in

catalysis.• How does destabilization of ES affect enzyme catalysis?• What factors influence enzymatic activity?• Have a general understanding of the 4 basic types of enzyme

catalysis discussed in this lecture.

In chemical reactions all reacting atoms or molecules pass through a state that is intermediate in structure between the reactant and product.

An Enzyme accelerates the reaction rate of a chemical reaction.

• For the rate of a reaction to be accelerated:1. Rate acceleration by an enzyme means that the

energy barrier between ES and EX ‡ must be smaller than the barrier between S and X‡

2. The enzyme must stabilize the transition-state complex (EX‡)

ΔGe‡ < ΔGu

‡

• KS=[E][S]/[ES]; KT=[E][X++]/[EX++] KT<KS

• Enzymatic rate enhancement is approximately equal to: ke/ku ~ KS/KT

How Does Destabilization of ES Affect Enzyme Catalysis?

Raising the energy of ES increases 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 ESb) destabilization of ES by

• strain• distortion• desolvation

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

Due to favorable interactions of S and AAs on enzyme


(a) Catalysis does not occur if ES and X‡ are equally stabilized. (b) Catalysis will occur if X‡ is stabilized more than ES.

Due to 1) Loss of entropy binding of S to E and 2) destabilization of ES due to: strain, distortion , desolvation

Favorable interactions of S and AAs on enzyme


(a) Formation of the ES complex results in entropy loss. The ES complex is a more highly ordered, low-entropy state for the substrate.


(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.


(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.

How Tightly Do Transition-State Analogs Bind to the Active Site?

• The affinity of the enzyme for the transition state may be 10 -20 to 10-26 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

• They bind do the enzyme more tightly than does the substrate but not as tightly as the natural substrate already in the transition state

Purine riboside inhibits adenosine deaminase. The hydrated form is an analog of the transition state of the reaction.

Transition-State Analogs and Drug Discovery

• 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 pesticide target

Tamiflu is a viral neuraminidase inhibitor

USP2a

Mdm2p53 ubiquitin Ligase

p53Tumor SuppressorP53

Ubiquitination

ubiquitin

Deubi

quiti

nates

Mdm

2ubiquitin

Mdm2p53 ubiquitin Ligase

ubiquitin

Degradation of p53Tumor Suppressor

USP2a expression in Cancer cells

1.00

10.00

100.00

USP2

a

Expression Purification and Crystallization of USP2a Core Domain

Expression: preformed in Ecoli strain Rosetta(DE3) pLysS

Purification:– Affinity chromatography (Ni column)– Size exclusion (Superdex 75)– Total yield (2 liters)≈ 60 mg USP2a. Crystallization:

Screened with/without Ubiquitin(8.5 kDa Boston Biochem)

48

33

195

142

96

71

2822

127.6

USP2a 40 kDa

100mM HEPES pH7.85mM DTT

5% PEG 4000

100mM Pipes pH7.85% PEG 4000

100mM HEPES pH7.85mM DTT, 10% Isopropanol 5% PEG 4000

Catalytic triad: 214-229 Cys-box;292-305 QDE-box;446-520 His-box218 oxyanion hole

• USP2a and ubiquitin have complementary shapes

• Of the 20 USP residues within 4 angstroms of the C-terminal penta-peptide 14 are conserved

• However, USP residues interacting with the ubiquitin core are poorly conserved

1.26Å Structure of USP2-ubiquitin Complex: Active Site and Vicinity

Active site residues shown on left (colored as in previous slide)• Two sites where interruption of ubiquitinin – USP2a interaction disrupts binding:

1. Large pocket in proximity of active site (C-terminus of ubiquitinin inserts here) 2. Second large pocket is visible on the right (area of Lys48 [colored in magenta] of

ubiquitinin)

USP2 is shown in green, ubiquitin is shown in cyan, water molecules are shown as blue spheres

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

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


• 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 pKa 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


Proton transfer can change a nucleophile into an electrophile, and vice versa.

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.


Water can often act as an acid or base at the active site through proton transfer with an assisting active-site residue.

Mechanisms of Catalysis• Enzymes facilitate formation of near-attack

complexes• Protein motions are essential to enzyme

catalysis• Covalent catalysis• General acid-base catalysis• Low-barrier hydrogen bonds• Metal ion catalysis

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 pKa values– Orientation of catalytic residues– Charge stabilization– Proton transfers via hydrogen tunneling

Enzymes facilitate formation of near-attack complexes

The energy separation between the NAC and the transition state is approximately the same in the presence and absence

of the enzyme.

Enzymes Facilitate Formation of NAC• Precise positioning

– Reacting atoms are in van der Waals contact – Angle resembling bond to be formed in

T-state• NACs are precursors to reaction transition states• Without enzyme, molecules adopt NAC ~0.0001%

of the time• NACs form in enzyme active sites 1%-70% of the

time

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

Protein Motions Are Essential to Enzyme Catalysis

• Proteins are constantly moving• 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

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.

Proteins are on the Move

Covalent Catalysis• Some enzymes derive much of their rate

acceleration from formation of covalent bonds between enzyme and substrate

• Amino acids side chains offer 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

Example 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.

General Acid-base CatalysisCatalysis 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

General Acid-base Catalysis

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

Mechanisms of Enzyme Action

Documents

Transcript of Mechanisms of Enzyme Action