Enzymes

Question 0

Free Energy / delta G

Answer 0

Free Energy change tells us whether a reaction can occur, not how fast it occurs •
Reaction occurs only if ΔG < 0. •
At equilibrium no net change takes place ΔG = 0. •
If ΔG > 0, energy must be supplied to produce reaction. •
ΔG only depends on reactants and products •
Mechanism is independent of ΔG. •
ΔG provides no information about the rate of reaction.

Question 1

Enzyme co-factors

Answer 1

Many enzymes need additional components to achieve the diversity necessary to catalyze the full range of reactions in biology.
Essentially a non protein required for protein to function

Apoenzyme + cofactor = holoenzyme
^enzyme
Without cofactor
Two-types: 1) coenzyme (organic) and 2) metals
Vitamins are precursors to ccoenzymes (not co-factor)

Question 2

Delta G not/ Standard free energy

Answer 2

ΔGo = Standard free energy (defined as the free energy when the substrate and product concentrations = 1 M)

Question 3

Standard free energy prime

Answer 3

ΔGo ́ = Biochemical standard free energy at pH 7.0

Question 4

What is the Effect of an Enzyme on a Reaction

Answer 4

An enzyme accelerates the rate of conversion of the substrate to product, but does not change the equilibrium constant (K eq ) between the substrate and product.

Question 5

Enzymes Catalyze by Stabilizing Transi8on States

Answer 5

Free energy G of a chemical reac8on can be ploZed over 8me (reac8on progress). •
Favorable reac8ons release energy (-­‐ΔG) as the reac8on proceeds. The free energy of the substrate > product. •
The free energy of ac8va8on for the transi8on state limits the progress of the reac8on. •
Enzymes act by reducing the free energy of the transi8on state. This is how they catalyze

Question 6

Active Sites of Enzymes

Answer 6

Active sites are usually clefts in the enzyme’s three dimensional structure. •
Water is usually excluded from these clefts. •
Active sites are only a small part of overall enzyme structure. • Substrates are bound to the enzyme through multiple weak interactions (3-12 kcal/mole or K eq of 10 -2 to 10 -8 ) that are precisely defined by the enzyme structure.
Ac8ve Site are Frequently Composed of Non-­‐con8guous Amino Acids

Question 7

Substrate Recognition by Enzymes: 2 models

Answer 7

Lock and Key – Complementary fit between the shapes of the enzyme’s active site and the substrate.

Induced Fit – Binding of the substrate induces a conformational change in the enzyme’s active site that promotes association.

Question 8

Catalytic Strategies Used by Enzymes

Answer 8

Covalent, acid base, metal ion catalysis
And
Catalysis by ApproximaEon and OrientaEon

Question 9

Acid base catalysis

Answer 9

A molecule in the ac8ve site beside water, such as amino acid in the enzyme, func8ons as a proton donor or acceptor during the reac8on. His57 in chymotrypsin is an example.

Question 10

Covalent catalysis

Answer 10

The ac8ve site of the enzyme possesses a reac8ve group, typically a nucleophile, that forms a covalent intermediate during the reac8on. Ser195 in chymotrypsin is an example.

Question 11

Metal ion catalysis

Answer 11

Metal ions can have several roles in enzyme catalysis:

a. They can func8on as an electrophilic catalyst by stabilizing a nega8ve charge in a reac8on intermediate.
b. Metal ions can also generate a nucleophile by increasing the acidity of a neighboring molecule, such as water.
c. Some metal ions can par8cipate in oxida8on/reduc8on reac8ons by altering their redox state.

Question 12

Catalysis by approximation and I orientation

Answer 12

Catalysis by ApproximaEon and OrientaEon – In enzyme-­‐catalyzed reac8ons involving two or more substrates, the ac8ve site brings the substrates together in an op8mal proximity and orienta8on for the reac8on to occur.
In liquid, due to brownian motion, interactions are random so this is important

Question 13

Common Features in Proteases – Enzymes that Hydrolyze Pep8de Bonds in Proteins

Answer 13

1. Activate H 2 O or other nucleophile
2. Polarize the peptide carbonyl group
3. Stabilize a tetrahedral intermediate

Question 14

Specificity of the Serine Protease Chymotrypsin

Answer 14

Chymotrypsin cleaves the peptide bond following the amino acids tryptophan, tyrosine, phenylalanine and methionine.

Question 15

Chymotrypsin and Other Proteases Use Covalent Catalysis in Hydrolyzing Pep8de Bonds

Answer 15

A. The pep8de bond in the protein substrate is broken, and the carboxyl component forms an acyl bond with the enzyme, yielding a species called the acyl-­‐enzyme intermediate.
B. A water molecule hydrolyzes the acyl bond in the acyl-­‐enzyme intermediate, regenera8ng the free enzyme.

Question 16

Michaelis-Menten Kinetics (Steady-State Kinetics)

Answer 16

E = enzyme, S = substrate, P = product •
ES = enzyme-substrate complex that is necessary for reaction • “Reaction is irreversible, once product is formed” •
Assumes [S]»[E] •
V 0 – Initial velocity of the enzyme catalyzed reaction
K m – The concentration of substrate at which the reaction rate is half of maximum rate (i.e., V 0 = 0.5V max ).
V max – maximum rate of the reaction at a given enzyme concentration.

Question 17

k cat : The Turnover Numbers

Answer 17

Turnover Number (k cat ) – The number of reacons catalyzed in the acve site per unit *me (e.g., per second, s -­‐1 ) when the enzyme is saturated with substrate ([S]&raquo_space; K m ).

Question 18

Comparing Enzyma*c Catalysis: k cat /K M

Answer 18

The cataly*c efficiency is defined as the value of k cat /K M . •

The table below illustrates that chymotrypsin prefers Phe as a substrate (has the highest k cat /K M value).

Question 19

Important Steady-State Kinetics Points (km and kcat)

Answer 19

K m ([S] when V max /2) : Provides an approximate idea of the binding “affinity” of the enzyme for the substrate, but K m has no information on rate. •
k cat : The turnover number tells how fast the enzyme- catalyzed reaction occurs, but only when the enzyme is saturated with substrate ([S]&raquo_space; K m ). It provides no information on substrate binding. •
k cat /K m : Catalytic efficiency allows a comparison of the activities of different enzymes, or the activities that a single enzyme displays toward different substrates.

Question 20

Enzyme and Temp/pH

Answer 20

Enzymes o[en exhibit opmal acvity at a temperature that is related to the biological environment in which they funcon.
Enzymes o[en display opmal acvity at a pH range that is consistent with the environmental condions in which they funcon.
For example, the protease pepsin funcons within the stomach, whereas chymotrypsin funcons in the small intesne.

Question 21

Enzyme Inhibitors

Answer 21

An inhibitor is a molecule that interferes with catalysis •
May affect K m or V max or both •
Inhibitors may be reversible or irreversible •
Irreversible inhibitors include“suicide inhibitors” •
QThere are 3 distinct classes of reversible inhibitors: - Competitive - Uncompetitive - Noncompetitive

Question 22

Reversible Enzyme Inhibi*on Compeve vs. Noncompeve vs. Uncompe**ve

Answer 22

Enzyme-­‐substrate (ES) complex B.
Compe??ve Inhibi?on: Inhibitor competes with the substrate for binding in the enzyme’s ac*ve site.
C. Uncompe??ve Inhibi?on: Inhibitor binds to the ES complex.
D. Noncompe??ve Inhibi?on: Inhibitor can bind to either the enzyme or the ES complex. It does not prevent the substrate from binding to the enzyme.

Question 23

Competitive Inhibitors Are Often Structurally Similar to Substrates for the Enzyme

Answer 23

Example: Dihydrofolate Reductase (DHFR) •
Methotrexate is a compe**ve inhibitor of the substrate tetrahydrofolate. • Methotrexate was one of the first an*-­‐cancer drugs developed.

Question 24

Competitive inhibitors (km vmax effects)

Answer 24

Increase the apparent K M but not the V max

The K i is the concentration of inhibitor that increases the Km by a factor of 2

Question 25

Uncompetitive inhibitors vmax and km

Answer 25

Uncompetitive Inhibitors Decrease the apparent V max and the K M

K i for the inhibitor is the concentration that decreases V max by a factor of 2

Question 26

Noncompetitive inhibitors vmax, km

Answer 26

Decreases the apparent V max but not the K M

K i for the inhibitor is the concentration that decreases V max by a factor of 2

Question 27

Comparing Types of Reversible Inhibitors km vmax

Answer 27

Competitive: Apparent Km increased; Vmax unaffected

Uncompetitive: Apparent Km and Vmax decreased

Noncompetitive: Apparent Vmax decreased; Km unaffected

Question 28

Irreversible Inhibitors

Answer 28

Modify the enzyme or cofactor by forming new covalent bonds • Irreversible inhibitors do not dissociate from the enzyme, unlike reversible inhibitors. •
Three types:
1) Group-­‐specific reagents
2) Affinity labels (reac*ve substrate analogs)
3) Suicide inhibitors (mechanism-­‐based inhibitors)

Question 29

Group Specific Reagents

Answer 29

React with the side chains of specific amino acids. •

Example: Diisopropylphosphofluoridate (DIPF) – forms covalent bonds with hydroxyl groups of reactive serines, such Ser195 in the active site of Chymotrypsin and the active site serine in Acetylcholinesterase.

Question 30

Affinity Labels

Answer 30

Affinity Labels •
Affinity Labels – Substrates can be chemically modified to generate specific covalent labeling reagents (i.e., reacve substrate analogs) that react with acve site residues in an enzyme. •
Example: Tosyl-­‐L-­‐phenylalanine chloromethyl ketone (TPCK) – A reacve substrate analog that forms a covalent bond with the His57 in the catalyc triad of chymotrypsin.

Question 31

Suicide inhibitor

Answer 31

Suicide Inhibitor (Mechanism-based Inhibitor) – A chemical modified substrate that begins to react with an enzyme like the substrate, but forms a highly reactive intermediate that covalently modifies an amino acid in the active site. •  
Example: Penicillin – a β-lactam antibiotic that inhibits the biosynthesis of the cell wall in bacteria (will be discussed in Lecture 8: Enzymes: Mechanisms of Drug Action).

Question 32

Allosteric Regulation of Enzyme Activity •

Answer 32

Allosteric Regulation – regulation of enzyme or protein activity by binding of a molecule at a site other than the active site (called the allosteric site). •
Conformational changes occur and are propagated between the allosteric site and active site, thus affecting activity. •
Allosteric enzymes exhibit Relaxed (R) and Tense (T) states. •
Example - aspartate transcarbamoylase (ATCase)

Question 33

Allosteric Enzymes are Regulated by the Products of the Pathways Under Their Control

Answer 33

The figure above represents a hypothecal metabolic pathway. A and E are the substrate and product of the pathway, whereas B, C, and D are intermediates. E 1 , E 2 , E 3 and E 4 are the four enzymes in the pathway. •
The product E binds to E 1 and inhibits the conversion of A to B, thus inhibing the enre pathway.
This phenomenon is referred to as feedback inhibion. •
Feedback inhibion prevents the accumulaon of E, as well as the accumulaon of the intermediates B, C, and D, when the concentraon of E is high in the cell. •
Feedback inhibion frequently occurs with the enzyme that catalyzes either the first or the rate-­‐liming step in the pathway.

Question 34

Aspartate Transcarbamoylase (ATCase)

Answer 34

ATCase is a multi-subunit enzyme that catalyzes the first committed step in the biosynthesis of pyrimidine nucleotides, such as cytidine triphosphate (CTP) •
The product of this biosynthetic pathway, CTP, inhibits ATCase through binding at a site outside of the active site.
This is an example of feedback inhibition. •
This alternative CTP binding site is referred to as an allosteric site.

Question 35

ATCase Shows Cooperativity

Answer 35

A plot of aspartate substrate concentraon vs. rate illustrates that the kinecs of ATCase do not obey a simple Michaelis Menten model. • The subunits of ATCase can exist in two different conformaons called the Tense (T) and Relaxed (R) states. •
The T state displays a lower binding affinity for substrate than the R state. •
Binding of substrate to the enzyme induces a conformaon change from the T to the R state, enhancing the enzyme’s binding affinity for addional molecules of substrate. •
This change in conformaon and substrate binding affinity is referred to as cooperavity and explains the sigmoidal kinec plot of ATCase.

Question 36

Basis for the Sigmoidal Kine*cs of ATCase

Answer 36

Black Curve: Sigmoidal kinec behavior of ATCase illustrates that it can exist in both the T and R states. •
T state: Displays a weak binding affinity for the substrate aspartate •
R state: Displays a strong binding affinity for aspartate •
Binding of aspartate results in conformaonal changes in ATCase, shi[ing the equilibrium from the T to the R state, which favors *ghter substrate binding.

Question 37

Effect of CTP and ATP on the Ac*vity of ATCase

Answer 37

Binding of CTP to ATCase favors the T state of the enzyme, resulting in a higher apparent K M value (weaker binding affinity) for the substrate aspartate.
The effect of CTP is an example of feedback inhibition.
Binding of ATP favors the R state of the ATCase, resulting in a lower apparent K M value (stronger binding affinity) for the substrate aspartate.

Question 38

Concerted Model for Allosteric Regula*on

Answer 38

R state infectious, all or nothing for subunits
In the concerted model, the enzyme exist only in the T state or the R state. •
In the absence of substrate, the enzyme exists mostly in the T state (A). •
As substrate concentraon rises, the equilibrium begins to shi[ from the T to R state (B and C). •
In the presence of high substrate concentraons, the enzyme exists predominantly in the R state (D).

Question 39

Sequential model allosteric

Answer 39

• In the absence of substrate, the enzyme exists in the T state. •
The binding of the substrate to one subunit alters the conformaon of that subunit from the T to the R state. •
The subunit bound to the substrate that is in the R state smulates the neighboring subunits that it contacts to begin changing their conformaon from the T to the R state, increasing their affinity for the substrate. •
The neighboring subunits bind substrate and change their conformaon to the R state, smulang other subunits to begin changing their conformaon from the T to the R state unl all subunits are in the R state and bound to substrate.

Question 40

Comparing Reversible inhibition

Answer 40

Compe&&ve – The substrate and inhibitor compete for binding in the acve site of the enzyme. Because they compete for binding, a higher concentraon of substrate is required to saturate the enzyme, increasing the K m value. At very high concentraons of substrate, the substrate outcompetes the inhibitor for binding to the enzyme and will saturate the enzyme to form the ES complex; thus, the V max value is unchanged. •
Uncompe&&ve – The inhibitor binds to the ES complex, forming an ESI complex. The formaon of the ESI complex decreases the concentraon of the ES complex in soluon which shiHs the equilibrium to form more ES complex, thus reduces the K m value. The ESI complex is enzymacally inacve and cannot form the product, reducing the V max value. •
Noncompe&&ve – The inhibitor can bind to the free enzyme to form an EI complex, or to the ES complex to form an EIS complex. Because the inhibitor does not interfere with the binding of the substrate to the enzyme, the K m value is unchanged. The EIS complex is enzymacally inacve and cannot form the product, reducing the V max value.

Question 41

Important properties of effective drugs

Answer 41

Drugs must be easily administered to paents -­‐ e.g., small tablets. • Should have minimal toxicity and side effects. •
Must survive in body long enough to reach the target(s) and have an effect. •
Must not modulate properes of molecules other than the targets •
Be cleared within a reasonable period of *me to diminish toxicity

Question 42

Two Approaches to Drug Discovery

Answer 42

Category (A): OHen includes natural products, such as aspirin, penicillin and quinine. Category (B): Include raonally designed drugs, such as HIV an-­‐ retrovirals.

Question 43

Half life of drug determines administration

Answer 43

The half-­‐life a drug in the body is determined by the rate that is eliminated by oxidaon, conjugaon to another molecule, and/or excre*on.

Question 44

Albumin Carries Lipophilic Drugs in the Blood

Answer 44

Serum albumin binds to many drugs (such as ibuprofen) and transports them throughout the body via the cardiovascular system.

Question 45

Metabolism of Drugs Alters Their Ac*vity

Answer 45

Oxidaon of ibuprofen by Cytochrome P450 increases its solubility, aiding in the drug’s excreon from the body.

Question 46

High Drug Levels Can Lead to Cytotoxicity

Answer 46

Example: Hepatotoxicity of Acetaminophen (Tylenol

Depletes glutathione (GSH), resul*ng in irreversible damage to the liver.

Question 47

Penicillin – The First Known Anbioc

Answer 47

First discovered by Alexander Flemming in 1928. •
Penicillin is secreted by the mold Penicillium notatum and kills bacteria, such as Staphylococcus Aureus. •
It was later shown that penicillin inhibits cell wall biosynthesis in bacteria.
Penicillin is the founding member of the β-­‐lactam family of anbiocs. • Anbiocs from this family share a β-­‐ lactam ring structure. • The “R” group represents different chemical groups that disnguish different β-­‐lactam anbio*cs.
Penicillin Inhibits Bacterial Cell Wall Synthesis (peptidoglycan)

Question 48

Mechanism of Glycopepde Transpepdase

Answer 48

Glycopepde transpepdase cleaves the pepde bond at the C-­‐terminus of the tetrapepde precursor and forms an acyl-­‐enzyme intermediate, similar to the mechanism of chymotrypsin.

In the second step of the reacon, glyco-­‐ pepde transpepdase transfers the acyl intermediate of the tetrapepde to the N-­‐ terminus of the pentaglycine bridge, forming a new pepde bond. This reacon differs from chymotrypsin in which a water molecule hydrolyzes the acyl-­‐enzyme intermediate.

Question 49

Structure of Penicillin

Answer 49

Thiazolidine ring (has sulfur)
Reactive bond is in square ring, between N and carbonyl carbon

Question 50

Penicillin is a Suicide Inhibitor of Glycopepde Transpepdase

Answer 50

Pencillin binds in the acve site of the glycopepde transpepdase by imitang the conformaon of the D-­‐Ala-­‐D-­‐Ala group of the substrate. • The serine in the acve site of the enzyme amacks the carbonyl carbon in the β-­‐lactam ring of penicillin, forming a stable acyl bond that inac*vates the enzyme.
This covalent complex is referred to as the penicilloyl-­‐enzyme complex.

Question 51

β-­‐lactamase – An Enzyme that Confers Resistance to β-­‐lactam Anbiocs by Hydrolyzing the β-­‐lactam Ring

Answer 51

β-­‐lactamase hydrolyzes the β-­‐lactam ring of penicillin and other anbiocs that have small R groups. β-­‐lactam anbiocs that have large R groups are not hydrolyzed efficiently by β-­‐lactamase (example: Cloxacillin).

Question 52

Clavulanic Acid

Answer 52

Overcoming β-­‐lactamase-­‐based Resistance

Isolated from Streptomyces clavuligerus •
Clavulanic acid contains a β-­‐lactam ring and resembles β–lactam drugs, but it is not an anbioc. •
Suicide inhibitor of β-­‐lactamase – binds in the enzyme’s acve site and covalently modifies the serine residue responsible for β-­‐lactam ring hydrolysis. •
Clavulanic acid is commonly used in combinaon with β-­‐lactam anbiocs (example: Augmen*n -­‐ amoxicillin plus clavulanic acid). • Bacterial strains with β-­‐lactamases resistant to clavulanic acid have emerged.

Question 53

HIV and An*retroviral Drugs

Answer 53

Human Immunodeficiency Virus (HIV) is a retrovirus (has an RNA-­‐ based genome) that infects human immune cells, including macrophages and helper T cells. •
HIV is the primary causave agent of Acquired Immune Deficiency Syndrome (AIDS). •
There are three primary targets for HIV anretroviral drugs:
HIV Reverse Transcriptase – Transcribes the virus’s single single-­‐stranded RNA genome into single-­‐stranded DNA.
HIV Integrase – Incorporates the transcribed viral DNA into the host’s genomic DNA.
HIV Protease – Cleaves the polyprotein, which is encoded by the viral DNA, into mature viral proteins.

Question 54

Impact of Anretroviral Drugs in Treang HIV 1987-­‐2000

Answer 54

HIV can rapidly mutate, leading to resistance to individual anretroviral drugs. •
Combina&on Therapy – Paents are treated with two or more anretroviral drugs that prevents HIV replicaon and muta*ons leading to drug resistance.

Question 55

Non-­‐steroidal An*-­‐inflammatory Drugs (NSAIDS) inhibit cyclooxygenase

Answer 55

Cyclooxygenase (COX) catalyzes the first step in prostaglandin synthesis by conver*ng arachidonic acid to prostaglandin H 2 in two steps. •

Question 56

Mechanism of COX Inhibi*on by Aspirin

Answer 56

Aspirin transfers its acetyl group to the hydroxyl group of Ser530 in COX.
The acetylaon of Ser530 obstructs the hydrophobic channel leading to the acve site, inhibing binding of the substrate arachidonic acid.
This reacon irreversibly inhibits COX.

Question 57

Cox and ibuprofen

Answer 57

Ibuprofen binds reversibly in the hydrophobic channel leading to the COX ac*ve site, blocking arachidonate binding. Naproxen also compe**vely inhibits COX by binding in this channel.