Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV …sites.fas.harvard.edu/~lsci1a/11-14notes.pdf · 2009. 6. 15. · Professor David Liu and Brian Tse, Life Sciences 1a

Professor David Liu and Brian Tse, Life Sciences 1a page 1

Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease

1. The function and structure of HIV proteasea. Introduction to proteasesb. Discovery of HIV proteasec. Overview of the three-dimensional structure of HIV protease

2. Chemical reactions and the energies driving thema. Amide bond cleavage: the reaction catalyzed by HIV proteaseb. Thermodynamics of a reaction and free energyc. Kinetics of a reaction and ΔG‡

d. Reaction energy diagramse. Transition states, intermediates, and how to draw them

3. How enzymes accelerate chemical reactions: the case of HIV proteasea. Catalysts alter a reaction’s kinetics, but not its thermodynamicsb. Chemical strategies behind enzyme catalysis

i. Proximity and orientation effectsii. Nucleophilicity and electrophilicityiii. Acid and base catalysis

4. The molecular basis of substrate specificitya. Trypsin substrate specificityb. HIV protease substrate specificity

Reaction energetics of amide bond hydrolysis

Let’s apply what we’ve learned about reaction thermodynamics and kinetics to the amidebond hydrolysis reaction catalyzed by HIV protease.

While it is sometimes possible to predict if a reaction under standard state conditions isfavorable (ΔG° < 0) or unfavorable (ΔG° > 0) with the assistance of several tables ofempirical and theoretical data, it is usually not possible to predict ΔG of a reaction simply byinspection, especially when the reaction does not take place under standard state conditions.The cleavage of an amide bond in water under typical physiological conditions, however, hasbeen empirically determined to be a favorable reaction.


O

O

OO

O

N

O

O

A

Proximity Effects on Reaction Rates

Rate at 1 M concentrations = 4 x 10-6 M/s

O

OO

N

O

O

B A B C

OOO

O

N

O

O

Rate = 0.8 M/s (200,000-fold faster!)

• Intramolecular reactions are much faster than intermolecular ones

A B

O

O

O

O

N

O

O

CA B

Chemical strategies behind enzyme catalysis: proximity and orientation effects

To effect the acceleration of chemical reactions, enzymes make use of a variety ofmolecular strategies, often with a remarkable effectiveness that is unmatched by analogouscatalysts developed in the laboratory. A major strategy is to organize the substrates within theactive site of the enzyme such that the reactants are much closer together than they would bein a typical solution. By enhancing the proximity of reactants, the effective concentration ofthe reactants (also called their effective molarity) can be dramatically increased. As a result,the reaction proceeds at a faster rate as if the substrate concentrations were dramaticallyincreased (recall that rate = k[substrate1][substrate2]…). We have already witnessed oneexample of this phenomenon early in the course. Recall that RNA cleavage occurs much fasterthan DNA cleavage because RNA’s 2’ hydroxyl group is positioned very close to a phosphategroup in the RNA strand. The resulting high effective molarity of the 2’ hydroxyl group and thephosphate group speeds their collision, which leads to RNA strand cleavage.

As an example of proximity effects in catalysis, consider the rates of the two reactionsshown above. The reaction at the top relies on the random collision between the twosubstrates to bring A and B close enough to react. In contrast, it’s much easier for A and B toencounter each other in the reaction at the bottom because A and B are already tetheredtogether. The effective concentration of A and B is much higher in the bottom reaction. Infact the rate constant of the top reaction has been measured to be ktop = 4 x 10-6 s-1M-1; incontrast, the rate constant of the bottom reaction is kbottom = 0.8 s-1. The effective molarity ofA and B in the bototm case is kbottom/ktop, or 200,000 M! In other words, A and B whentethered in close proximity react at the same rate as that of the untethered reaction when theconcentration of A or B is an impossibly high 200,000 M. Enzymes make use of the proximityeffect, often with similarly dramatic results, by confining reactants within the relatively smallactive site of the enzyme.


Orientation Effects

A B

A B

A B

Increasing rateof reaction

• The fewer nonproductive ways two groups can be oriented,the faster they will react

A

B

A

B

A

B

(assume A and B reactupon sideways collision)

A related but distinct strategy used by enzymes to accelerate chemical reactions is orientingsubstrates into a maximally reactive conformation. Simply confining two substrates to a smallseparation distance does not guarantee an increase in reaction rate or effective concentration,because in order for two substrates to react they usually must achieve a specific relativeorientation as you will learn in detail when you take organic chemistry. In the example above,it is not sufficient to simply tether A and B together; instead, a requirement of their reaction isto orient A and B such that one of the oxygen atoms in A is properly aligned with the carbonatom in B. Many enzymes catalyze reactions not only by holding substrates close together, butalso by forcing the substrates into an optimal orientation to lower the activation energy neededto reach the transition state.


Proximity and Orientation Effectsin HIV Protease

O

NH

R1HN

R2O O

HN

O

O

HN

R2O

HOH

NH

R1

O

HNH

+ H+

protein substrate

“attacking” water

HIV proteasebackbone

(Ile 50 and Ile 50')

• The enzyme’s activesite holds substrates(protein & water) inclose proximity

O C

N H

• HIV protease useshydrogen bonds toorient substratesproductively

H

O

H

N

O

HN

O

H

An examination of the active site structure of HIV protease reveals that this enzyme usesboth proximity and orientation effects to accelerate the rate of amide bond hydrolysis. Thetwo substrates of the amide hydrolysis reaction, water and a polyprotein, are held in closeproximity within the active site of the protein. In addition, the water molecule and proteinsubstrate are oriented precisely such that the electrons on the oxygen atom of water are well-positioned to form a bond with the carbon atom of the amide bond being cleaved in thepolyprotein substrate. HIV protease positions the two substrates by establishing a series ofhydrogen bonds and hydrophobic contacts between the enzyme and the substrates.Specifically, the amide –NH– groups of Ile 50 and Ile 50’ (the same amino acid in each of thetwo monomers of the dimeric enzyme) in HIV protease serve as hydrogen bond donors to theoxygen atom of the water molecule.


A Network of InteractionsPrecisely Positions the

Substrates of HIV Protease

Note: structure isof an inactiveAsn 25 mutant ofHIV proteasecomplexed witha substrate.

A number of other amino acid amide groups and side chains from the enzyme (includingAsp 25, Gly 27, Asp 29, and Gly 48) form hydrogen bonds with the amide backbone groups ofthe polyprotein substrate. Collectively, this network of interactions positions the bound watermolecule and the polyprotein into a conformation that is well suited for amide hydrolysis.


Nucleophiles and Electrophiles

Nu–E+Electrophile

electron-deficient,“likes electrons”

NE

Nucleophileelectron-rich,“likes nuclei”

• Nucleophilicity and electrophilicity are kinetic parameters• The more nucleophilic the nucleophile, or electrophilic the

electrophile, the faster the reaction (by definition!)

bond-formingreaction

u

Chemical strategies behind enzyme catalysis: nucleophilicity and electrophilicity

In addition to proximity and orientation effects, enzymes also catalyze reactions byproviding proton donors (acids) and proton acceptors (bases) at precise locations in the activesite. In the laboratory, chemists can accelerate many reactions by adding acid or base tolower or raise the pH of the reaction solution. These pH changes typically accelerate reactionsby altering the nucleophilicity or electrophilicity of the reactants.

As its name implies, nucleophilicity (“the degree of nucleus loving”) is a measure of howquickly an atom is able to form a covalent bond with an electron-deficient atom. Highlynucleophilic groups— which are electron-rich groups— are able to form this bond faster thanweak nucleophiles. Nucleophilicity is therefore a kinetic parameter— it reflects the rate ofbond formation between a nucleophile and an electron-deficient atom, rather than thethermodynamic property of the amount of energy that is released as a result of the bondformation.


Factors Governing Nucleophilicity

1) More basic molecules tend to be more nucleophilicwhen the nucleophilic atoms are of comparable size:

HO

H

Poor nucleophilepKa of conjugate acid = –1.5

Good nucleophilepKa of conjugate acid = 15.5

OH

2) Larger atoms (those lower on the periodic table) makebetter nucleophiles:

Weaker nucleophile Stronger nucleophileHS

HHO

H

Although nucleophiles must have at least one lone pair of electrons, they can vary widely intheir nucleophilicity. The best nucleophiles tend to be atoms with an excess of electrons. As ageneral rule of thumb, when comparing two atoms of similar size (such as among N, O, or F),the atom in the more basic (higher pKa) group is the more nucleophilic atom. Therefore,hydroxide anion (–OH), which in the protonated form (H2O) has a pKa of 15.5, is a much betternucleophile than water, which in the protonated form (H3O

+) has a pKa of -1.5. Likewise,ammonia (NH3) (pKa when protonated = 10) is a better nucleophile than water. In addition tousing basicity as a guide to judging nucleophilicity, larger atoms (such as those found in alower row of the periodic table) are better nucleophiles than similarly charged but smalleratoms. For example, H2S is a better nucleophile than H2O because sulfur is one row belowoxygen in the periodic table.


Electrophiles in the Molecules of Life

Peptide hydrolysis

• The most common electrophiles in the chemistry of life areC=O and P=O

• Groups that are more electron-poor are more electrophilicand therefore react more quickly with nucleophiles

O

OO

P

O

O

O

O

NH

Translation DNA polymerizationDNA hydrolysis

Protein phosphorylation

Conversely, electrophilicity is the measure of how quickly an electron-deficient atom is ableto form a covalent bond with an incoming nucleophile. Therefore, like nucleophilicity,electrophilicity is a kinetic parameter. In the chemical reactions of life, electrophilic atoms aremost often carbon or phosphorus atoms that are doubly bonded to oxygen atoms. Those ofyou who will take organic chemistry will learn in much more detail the factors that determineelectrophilicity. For the purposes of this course, however, we will focus on C=O and P=Ogroups as electrophiles. As you might expect, those atoms attached to groups that can mostreadily accept electrons (including groups made of electronegative atoms and groups thatcontain double or triple bonds) are the most electrophilic and make the best electrophiles.


Acids and Bases Can EnhanceElectrophilicity and Nucleophilicity

HO

HO

H

Better nucleophile

more electron-richBASE

:OH-

O

HN

Better electrophile

more electron-poor

ACID

H+

O

HN

H

Chemical strategies behind enzyme catalysis: acid and base catalysis

The protonation or deprotonation of a group can profoundly change its nucleophilicity orelectrophilicity. As we have already seen in the case of water, removing a proton from a grouptypically increases its electron richness, and therefore its nucleophilicity. Conversely, adding aproton to a group typically makes that group more readily accept electrons, thereby increasingits electrophilicity. For example, protonating the carbonyl oxygen atom in an amide results in adramatic increase in the ability of the amide to accept an incoming nucleophile.


O

HN

H

OH O

HN

O H

H

Base Catalysis of Amide Hydrolysis

Weaker nucleophile:slower reaction,

higher ΔG‡

‡

!+!"

O

HN

H

O

‡O

HN

O H!"H2O + base

Better nucleophile:faster reaction,

lower ΔG‡

δ−

• Base can accelerate amide hydrolysis by deprotonatingwater, increasing its nucleophilicity

Armed with this knowledge, you can now appreciate that neutral water is only very weaklynucleophilic, but deprotonated water (the hydroxide anion) is reactive enough to initiate amidebond hydrolysis. A chemist might speed up the rate of amide bond hydrolysis in a test tube byadding base in order to deprotonate water and thereby convert it into a much betternucleophile. Although enzymes in general cannot change the pH of an entire cell, they cansimilarly position an acidic or basic group at just the right location to greatly lower ΔG‡. Mostcommonly, these acids and bases are the side chains of acidic and basic amino acids describedin our earlier lectures on proteins.

Even though you can draw a reasonable transition state structure for each of the casesshown above, it is not obvious simply from inspecting the structures of these proposedtransition states which would represent a larger ΔG‡ value. Instead, you should reason that (i)hydroxide anion is a better nucleophile than water for the reasons discussed above; (ii)hydroxide anion’s superior nucleophilicity means that— by definition— the rate at whichhydroxide anion forms a bond with the amide carbonyl is higher than the rate at which waterforms a bond with the amide carbonyl; and (iii) the faster rate must correspond to a lower ΔG‡value.


O

HN

O

O

H

O

OH

O HO

HN

O

O

H

O

O

O H

H

O

HN

O

O

H

O

OH

OH

Base Catalysis by HIV Protease

‡

Asp 25of HIV protease

!" δ−

Asp 25of HIV protease

• HIV protease precisely positions Asp 25 to serve as a baseto deprotonate water

• The enhanced nucleophilicity of deprotonated wateraccelerates amide hydrolysis

deprotonationof water

HIV protease takes advantage of both acid and base catalysis. Asp 25, one of the criticalcatalytic aspartate residues in HIV protease, in its deprotonated form serves as a base todeprotonate the water molecule during the first step of the enzyme-catalyzed reaction. Thedeprotonated water is now a much better nucleophile and can attack the amide carbonyl groupof the substrate much faster than a neutral water molecule. Therefore, base catalysis by HIVprotease’s Asp 25 side chain greatly enhances the nucleophilicity of water and therefore itsability to initiate amide bond hydrolysis.


O

HN

O H

H

O

HN

H

OH

Acid Catalysis of Amide Hydrolysis‡

!"δ+

Weaker electrophile:slower reaction,

higher ΔG‡

O

HN

H

OHH

O

HN

O H

H

H

‡

δ+

Stronger electrophile:faster reaction,

lower ΔG‡

• Acid can accelerate amide hydrolysis by protonating theamide oxygen, increasing its electrophilicity

δ+

amide+

acid

During this attack by deprotonated water, an excess of electron density builds up on theoxygen atom of the amide group in the substrate. Without a nearby proton to neutralize thisdeveloping oxygen anion (“oxyanion”), the resulting transition state would be quite unstable(ΔG‡ would be high) and the resulting rate of amide hydrolysis would be slow. In otherwords, a neutral amide is not electrophilic enough to enable the nucleophile to efficientlyform a bond. In the test tube, a chemist could add acid to the reaction to protonate theoxyanion, increasing the amide’s electrophilicity and stabilizing the transition state.


O

HN

O

O

O

OH

O HHO

HN

O

O

H

O

O

O H

H

Acid Catalysis by HIV Protease

‡O

HN

O

O

H

O

OH

O H

Asp 25’of HIV

protease

!"δ−

Asp 25’of HIV

protease

• HIV protease precisely positions Asp 25’ to serve as anacid to protonate the substrate amide, increasing itselectrophilicity and accelerating amide hydrolysis

protonationof amide

Likewise, HIV protease uses acid catalysis to provide a proton to neutralize the formingoxyanion in the enzyme’s active site and therefore increase the substrate’s electrophilicity. Asp25’, the other catalytic Asp residue, is protonated as the carboxylic acid in the active form ofHIV protease. As the oxyanion on the substrate amide is being formed, the proton from Asp25’ is transferred to the oxyanion, stabilizing the transition state of the hydrolysis reaction.HIV protease therefore uses acid catalysis to stabilize the oxyanion that forms when waterattacks the substrate’s amide group.


Enzymes Can Catalyze Reactions in WaysThat Simple Acids and Bases Cannot

O

HN

H

OH

ACIDH +

BASE

:OH-

How can an acid and basesimultaneously catalyze a

single reaction? enzyme acid

O

HN

O

O

H

O

OH

OH

enzyme base

• Enzymes can simultaneously use acidic and basic groupsthat, in a flask, would wander and neutralize each other

You may realize based on the above discussion that HIV protease can catalyze amidehydrolysis in ways that a scientist working with test-tube reagents cannot. It would beimpossible for a scientist to simultaneously use acid catalysis and base catalysis in the samesolution by adding both a simple acid and a simple base to the same reaction, since the acidand base would rapidly neutralize each other. In contrast, an enzyme can precisely tailor itsactive site so that one region of the active site presents an acidic group to enhanceelectrophilicity, while the other region presents a basic group to enhance nucleophilicity. Thepositions and environments of of the acidic and basic groups of the enzyme are fixed such thatthey cannot neutralize each other. As a result of strategies such as simultaneous acid andbase catalysis, enzymes can achieve rate accelerations that in many cases remain unmatchedby manmade catalysts.


HIV Protease Active Site in Action

Asp 25’

Ile 50’ attackingwater

substrate

Note: locations of hydrogen atoms are usually inferred (and often not shown)

H

Asp 25


O

HN

O

O

H

O

OH

O H

HIV Protease Catalysis Summary

O

HN

O

O

H

O

OH

OH

‡O N

O

O

H

O

OH

O

H

H

O

O

O

OH

O

HN

O HH

Acidcatalysis

Basecatalysis

O

O

O

O

H

O

O

H

N

H

H

δ−δ−

O

HN

O

O

O

OH

O HH

‡

Acidcatalysis

Basecatalysis

δ−δ−

Tetrahedralintermediate

Asp 25’ Asp 25

Proximity andorientation effects

Integrating the above analysis forms the picture shown here for how HIV proteaseaccelerates the rate of amide bond cleavage. Asp 25 serves as a catalytic base to deprotonatewater. The resulting highly nucleophilic hydroxide ion attacks an amide bond in the substrate.The oxyanion that begins to develop on the way to the intermediate is stabilized by Asp 25’serving as a catalytic acid to protonate the forming oxyanion, increasing the amide’selectrophilicity. After the formation of the tetrahedral intermediate), the catalytic Asp residuesreverse their roles to complete the reaction. One of the hydroxyl groups in the tetrahedralintermediate is deprotonated by a catalytic aspartate (base catalysis), and the resulting anionicintermediate collapses. Instead of ejecting a hydroxide ion (which would represent exactly thereverse of the reaction thus far), the collapse of the tetrahedral intermediate causes the —NHgroup to break away. The departure of the —NH group (which in the absence of a catalystwould be negatively charged) is assisted by picking up a proton from a catalytic aspartic acidresidue (acid catalysis). Therefore, a series of base-catalyzed and acid-catalyzed stepstogether with proximity and orientation effects collectively enable HIV protease to mediate theefficient hydrolysis of an otherwise very stable amide bond.

You may wonder how the tetrahedral intermediate “knows” to collapse to break the C-Nbond instead of breaking a C-O bond. The answer is that the intermediate collapses in bothways— but breaking the C-O bond simply regenerates the starting materials for the reaction,and we are back to where we started. This principle of only one out of several mechanisticpossibilities leading to a productive reaction step is a very common principle underlying thechemistry of life. Even if the productive step takes place only a small fraction of the time,there is a net flow of starting materials to products.


Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease

1. The function and structure of HIV proteasea. Introduction to proteasesb. Discovery of HIV proteasec. Overview of the three-dimensional structure of HIV protease

2. Chemical reactions and the energies driving thema. Amide bond cleavage: the reaction catalyzed by HIV proteaseb. Thermodynamics of a reaction and free energyc. Kinetics of a reaction and ΔG‡

d. Reaction energy diagramse. Transition states, intermediates, and how to draw them

3. How enzymes accelerate chemical reactions: the case of HIV proteasea. Catalysts alter a reaction’s kinetics, but not its thermodynamicsb. Chemical strategies behind enzyme catalysis

i. Proximity and orientation effectsii. Nucleophilicity and electrophilicityiii. Acid and base catalysis

4. The molecular basis of substrate specificitya. Trypsin substrate specificityb. HIV protease substrate specificity


O

NH

P3HN

P4O O

HN

O

NH

P1

P2 O

HN

O

NH

P2'

P1' O

HN

O

NH

P4'

P3' O

HN

Protease Substrate Specificity

To C-terminal endof substrate

To N-terminal endof substrate

Scissilebond

S3 S1

S4 S2

S2’ S4’

S3’S1’

Enzyme specificitypockets recognize the

specific amino acidresidues surrounding the

bond to be hydrolyzed

4. Molecular basis of substrate specificity

For most enzymes, merely accelerating the rate of a chemical reaction that would otherwiseproceed too slowly is not sufficient to be useful to a living system. Most enzymes must also bequite selective in the substrates that they convert to products to avoid interfering with otheressential life processes. There is nothing about the catalytic strategy of HIV protease thatwould explain how it is able to exclusively cleave the correct amide bonds out of a vast numberof possibilities. Instead, this specificity arises from many favorable interactions— mostlyoutside of the active site— between HIV protease and its protein substrates.

Trypsin substrate specificity

Let’s begin to understand the origins of an enzyme’s substrate specificity by examiningtrypsin, one of your digestive proteases. The major biological roles of trypsin in your body areto catalyze the hydrolysis of protein consumed as food. If trypsin catalyzed the hydrolysis ofany peptide bond (i.e., if trypsin had no substrate specificity) then it would not only digest theproteins in your food but would also destroy other trypsin molecules as well as other importantdigestive enzymes. Fortunately, trypsin will only cleave proteins after exposed lysine orarginine residues. This simple substrate specificity helps trypsin avoid the haphazardhydrolysis of necessary protein enzymes in your digestive system.

Before we reveal the molecular basis of this specificity, we must learn a few simple terms.The amide bond being cleaved by a protease is called the scissile bond. The amino acids inthe substrate that extend from the scissile bond toward the N-terminus of the substrate aredesignated P1, P2, P3, etc. Likewise, the amino acids that extend from the scissile bondtoward the C-terminus of the substrate are called P1’, P2’, P3’, etc. The scissile bond istherefore the amide bond connecting the P1 and P1’ amino acids.


Trypsin: A Digestive Protease that CleavesSubstrates Containing Lys or Arg

trypsin activesite residues

enzymeAsp189

S1

scissile bond

substrateLys P1

substrate peptide

trypsinenzyme

+–

Each of the substrate’s amino acid side chains interacts with a binding pocket in theprotease that is formed from the conformations of the many residues that make up theenzyme. These binding pockets are designated S1, S2, S3, S1’, S2’, S3’, etc. In the case oftrypsin, P1 must be Lys or Arg in order for the enzyme to catalyze efficient peptide bondhydrolysis.

What is the molecular basis of trypsin’s P1 substrate specificity? Fortunately the three-dimensional structure of trypsin, like that of HIV protease, has been revealed in great detail.An examination of this structure reveals that the trypsin’s S1 substrate binding pocket containsa negatively charged aspartic acid (Asp) 189 residue.


Basis of Trypsin Substrate Specificity

Asp 189

O O

NH2H2N

NH

NH

O

HN

O O

NH

O

HN

H3N

LysP1

ArgP1

Asp 189S1 S1

• The presence of anionic Asp 189 in the S1 site causes astrong preference for P1 to be a cationic Lys or Arg

The resulting anionic S1 site results in a strong electrostatic preference for P1 to bepositively charged. Since Lys and Arg are fully positively charged amino acids underphysiological conditions, and because the length and shape of Lys and Arg fit the S1 bindingpocket’s length and shape, the presence of Asp 189 in the S1 binding pocket confers theobserved [P1 = Lys or Arg] substrate specificity. Complementary protein-substrate interactionssuch as this one are major determinants of enzyme substrate specificities.


HIV Protease-Substrate Interactions

Animation rendered by Brian Tse

HIV protease substrate specificity

As a more complex example of protease substrate specificity, let’s consider HIV proteaseonce again. There are roughly 1,500 peptide amide linkages in the Gag and Gag-Pro-Polpolyproteins, yet HIV protease cleaves only 10 of these amide bonds.

Understanding the basis of HIV protease’s substrate specificity is particularly importantbecause inhibitors of HIV protease including those currently used to treat AIDS must beaccepted by HIV protease and bind to the enzyme. Most enzyme inhibitors, including those ofHIV protease, are molecules that mimic the shape and chemical properties of the substrate ortransition state but cannot be converted into a product for chemical reasons. To be maximallyeffective, an enzyme inhibitor should ideally bind to the enzyme with an affinity that is greaterthan that of the natural substrate. Such an inhibitor can sequester the vast majority of theenzyme’s active sites in a cell, preventing the enzyme from efficiently processing its naturalsubstrate.


The HIV Protease ActiveSite: A Closer Look

Note: structure isof an inactiveAsn 25 mutant ofHIV proteasecomplexed witha substrate.

HIV protease substrate specificity

As a more complex example of protease substrate specificity, let’s consider HIV proteaseonce again. There are roughly 1,500 peptide amide linkages in the Gag and Gag-Pro-Polpolyproteins, yet HIV protease cleaves only 10 of these amide bonds.

Understanding the basis of HIV protease’s substrate specificity is particularly importantbecause inhibitors of HIV protease including those currently used to treat AIDS must beaccepted by HIV protease and bind to the enzyme. Most enzyme inhibitors, including those ofHIV protease, are molecules that mimic the shape and chemical properties of the substrate ortransition state but cannot be converted into a product for chemical reasons. To be maximallyeffective, an enzyme inhibitor should ideally bind to the enzyme with an affinity that is greaterthan that of the natural substrate. Such an inhibitor can sequester the vast majority of theenzyme’s active sites in a cell, preventing the enzyme from efficiently processing its naturalsubstrate.


HIV Protease Substrate Selectivity

• HIV protease recognizes more substrate amino acids thantrypsin, but without a strong preference at any one position

The 10 sites cleaved by HIV protease:

Key: MA = matrix; CA = capsid; NC = nucleocapsid; TF = trans frame peptide; PR = protease;AutoP = autoproteolysis (self-cleaving) site; RT = reverse transcriptase; RH = RNAse H; integrase = IN

Unlike trypsin, HIV protease interacts with at least seven amino acids in the substrate. Aswe will describe in detail in the next lecture, these binding pocket-substrate interactions aretypically a mixture of hydrophobic, hydrogen bonding, and ionic interactions. Despitecontaining a much larger number of amino acid binding pockets than trypsin, HIV proteaseactually exhibits a greater tolerance for different amino acids at any one of these positionsthan trypsin’s strong preference for a Lys or Arg in the P1 position. HIV protease is therefore abit unusual in that its substrate specificity is quite broad for any one amino acid position. Dueto the large number of substrate binding pockets, however, the overall specificity of HIVprotease is quite narrow because all of these pockets contribute to the overall specificity.


O

NH

HN

O O

NO

NH

O

HN

HN

N NO

NH

HN

O

NH

H

HOH

H

H

H

H

HIV Protease Specificity: P1 and P3

Asn 25’

• HIV protease’sstructure can explainsome aspects of itssubstrate specificity

• P3 = polar orcharged; S3 containsa bound water

Arg P3

waterS3

Gly 49S1

Leu P1

• P1= large andhydrophobic;complementsS1 = Gly 49

Despite its ability to tolerate a variety of amino acids at most substrate positions, HIVprotease makes a number of substrate interactions that are representative of the ways inwhich enzymes recognize their substrates. Shown here are some of the interactions betweenHIV protease and the P1 and P3 residues of one of its natural substrates. You can see that inthe structure of the enzyme is well matched to bind the amino acids that commonly occur atthe corresponding substrate position. For example, the P1 amino acid is usually large andhydrophobic, and the P1 side chain fits into a water-excluded cavity in the enzyme’s active sitecreated by the absence of a side chain at Gly 49. Likewise, the preference of P3 for a polar orcharged amino acid likely reflects the bound water molecule in the enzyme’s P3 binding pocketthat can make favorable hydrogen bonds with polar side chains of amino acids such as Arg.Collectively, many such interactions define the substrate specificity of HIV protease.

The interactions that enzymes make with their substrates to determine substrate specificityare similar to those that enzymes make with transitions states to lower ΔG‡ and acceleratechemical reactions. If you take another look at the protease residues involved in proximity andorientation catalysis as well as base and acid catalysis you will find many of the same chemicalthemes. Conveying an intuitive understanding of protein-substrate and protein-transition stateinteractions is a major aspect of this course. Indeed, such an understanding recently enabledscientists to develop a major new class of HIV therapeutics that have had a major impact onextending the life of patients afflicted with AIDS. These small-molecule drugs, the HIVprotease inhibitors, and the principles underlying their development are the subject of the nextlectures in this course.


Key Points: HIV Protease & Enzyme Catalysis• HIV protease catalyzes polyprotein amide bond hydrolysis• Thermodynamics reflect the difference in energy between

reactants and products, as measured by ΔG°rxn

• Kinetics reflect reaction rates, determined by ΔG‡

• Enzymes lower ΔG‡ by using a variety of chemical strategiesto create a precise transition state-stabilizing active siteenvironment

• Enzymes use proximity and orientation effects to increasethe concentration of substrates, increasing rates of reactions

• Enzymes use acid and base catalysis to enhance thenucleophilicity or electrophilicity of reactants

• Specificity arises from protein-substrate interactions

Documents

Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV …sites.fas.harvard.edu/~lsci1a/11-14notes.pdf · 2009. 6. 15. · Professor David Liu and Brian Tse, Life Sciences 1a