Page 1 Proximity Effects on Reaction Rates

Home , Enzyme catalysis

Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease 1. The function and structure of HIV protease a. Introduction to proteases b. Discovery of HIV protease c. Overview of the three-dimensional structure of HIV protease 2. Chemical reactions and the energies driving them a. Amide bond cleavage: the reaction catalyzed by HIV protease b. Thermodynamics of a reaction and free energy c. Kinetics of a reaction and ΔG‡ d. Reaction energy diagrams e. Transition states, intermediates, and how to draw them 3. How enzymes accelerate chemical reactions: the case of HIV protease a. Catalysts alter a reaction’s kinetics, but not its thermodynamics b. Chemical strategies behind enzyme catalysis i. Proximity and orientation effects ii. Nucleophilicity and electrophilicity iii. Acid and base catalysis 4. The molecular basis of substrate specificity a. Trypsin substrate specificity b. HIV protease substrate specificity

Reaction energetics of amide bond hydrolysis

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

While it is sometimes possible to predict if a reaction under standard state conditions is favorable (ΔG° < 0) or unfavorable (ΔG° > 0) with the assistance of several tables of empirical and theoretical data, it is usually not possible to predict ΔG of a reaction simply by inspection, 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, has been empirically determined to be a favorable reaction.

Professor David Liu and Brian Tse, Life Sciences 1a page 1 Proximity Effects on Reaction Rates

O O N O O O O O N O O O O O A B A B C Rate at 1 M concentrations = 4 x 10-6 M/s

O O O N O O N O O O O O O O

A B A B C

Rate = 0.8 M/s (200,000-fold faster!) • Intramolecular reactions are much faster than intermolecular ones

Chemical strategies behind enzyme catalysis: proximity and orientation effects

To effect the acceleration of chemical reactions, enzymes make use of a variety of molecular strategies, often with a remarkable effectiveness that is unmatched by analogous catalysts developed in the laboratory. A major strategy is to organize the substrates within the active site of the enzyme such that the reactants are much closer together than they would be in a typical solution. By enhancing the proximity of reactants, the effective concentration of the 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 dramatically increased (recall that rate = k[substrate1][substrate2]…). We have already witnessed one example of this phenomenon early in the course. Recall that RNA cleavage occurs much faster than DNA cleavage because RNA’s 2’ hydroxyl group is positioned very close to a phosphate group in the RNA strand. The resulting high effective molarity of the 2’ hydroxyl group and the phosphate group speeds their collision, which leads to RNA strand cleavage.

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

Professor David Liu and Brian Tse, Life Sciences 1a page 2 Orientation Effects B B

A B A A B Increasing rate A B A of reaction

(assume A and B react upon sideways collision) A B • The fewer nonproductive ways two groups can be oriented, the faster they will react

A related but distinct strategy used by enzymes to accelerate chemical reactions is orienting substrates into a maximally reactive conformation. Simply confining two substrates to a small separation 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 relative orientation 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 is to orient A and B such that one of the oxygen atoms in A is properly aligned with the carbon atom in B. Many enzymes catalyze reactions not only by holding substrates close together, but also by forcing the substrates into an optimal orientation to lower the activation energy needed to reach the transition state.

Professor David Liu and Brian Tse, Life Sciences 1a page 3 Proximity and Orientation Effects in HIV Protease

O R1 O R H H H 1 H N N O N H H N N + H H O + N H H O R2 O O R2 O

O O HIV protease • HIV protease uses N N H backbone hydrogen bonds to H (Ile 50 and Ile 50') orient substrates productively O H H “attacking” water O C • The enzyme’s active N H site holds substrates (protein & water) in protein substrate close proximity

An examination of the active site structure of HIV protease reveals that this enzyme uses both proximity and orientation effects to accelerate the rate of amide bond hydrolysis. The two substrates of the amide hydrolysis reaction, water and a polyprotein, are held in close proximity within the active site of the protein. In addition, the water molecule and protein substrate 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 the polyprotein substrate. HIV protease positions the two substrates by establishing a series of hydrogen 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 the two monomers of the dimeric enzyme) in HIV protease serve as hydrogen bond donors to the oxygen atom of the water molecule.

Professor David Liu and Brian Tse, Life Sciences 1a page 4 A Network of Interactions Precisely Positions the Substrates of HIV Protease

Note: structure is of an inactive Asn 25 mutant of HIV protease complexed with a substrate.

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

Professor David Liu and Brian Tse, Life Sciences 1a page 5 Nucleophiles and Electrophiles

Electrophile + – Nucleophile electron-deﬁcient, electron-rich, “likes electrons” E Nu “likes nuclei” bond-forming reaction E Nu

• Nucleophilicity and electrophilicity are kinetic parameters • The more nucleophilic the nucleophile, or electrophilic the electrophile, the faster the reaction (by deﬁnition!)

Chemical strategies behind enzyme catalysis: nucleophilicity and electrophilicity

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

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

Professor David Liu and Brian Tse, Life Sciences 1a page 6 Factors Governing Nucleophilicity

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

O O H H H Poor nucleophile Good nucleophile

pKa of conjugate acid = –1.5 pKa of conjugate acid = 15.5

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

O S H H H H Weaker nucleophile Stronger nucleophile

Although nucleophiles must have at least one lone pair of electrons, they can vary widely in their nucleophilicity. The best nucleophiles tend to be atoms with an excess of electrons. As a general 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 better + nucleophile 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 to using basicity as a guide to judging nucleophilicity, larger atoms (such as those found in a lower row of the periodic table) are better nucleophiles than similarly charged but smaller

atoms. For example, H2S is a better nucleophile than H2O because sulfur is one row below oxygen in the periodic table.

Professor David Liu and Brian Tse, Life Sciences 1a page 7 Electrophiles in the Molecules of Life

O O O P O N O O H O Peptide hydrolysis Translation DNA polymerization DNA hydrolysis Protein phosphorylation

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

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

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

Professor David Liu and Brian Tse, Life Sciences 1a page 8 Acids and Bases Can Enhance Electrophilicity and Nucleophilicity

more electron-rich BASE - O :OH O H H H Better nucleophile

H H N ACID N H+ more electron-poor O O H Better electrophile

Chemical strategies behind enzyme catalysis: acid and base catalysis

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

Professor David Liu and Brian Tse, Life Sciences 1a page 9 Base Catalysis of Amide Hydrolysis

H N H ‡ Weaker nucleophile: N slower reaction, O ‡ H " O O H higher G O ! Δ H + H !

H N Better nucleophile: H ‡ N faster reaction, O ‡ O " O O H lower ΔG H2O + base ! − H δ

• Base can accelerate amide hydrolysis by deprotonating water, increasing its nucleophilicity

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

Even though you can draw a reasonable transition state structure for each of the cases shown above, it is not obvious simply from inspecting the structures of these proposed transition 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 which hydroxide anion forms a bond with the amide carbonyl is higher than the rate at which water forms a bond with the amide carbonyl; and (iii) the faster rate must correspond to a lower ΔG‡ value.

Professor David Liu and Brian Tse, Life Sciences 1a page 10 Base Catalysis by HIV Protease

H N H N H N ‡ O deprotonation O H of water " O ! O O H O H δ− H H H H O O O O H O H O O O O O O O

Asp 25 Asp 25 of HIV protease of HIV protease • HIV protease precisely positions Asp 25 to serve as a base to deprotonate water • The enhanced nucleophilicity of deprotonated water accelerates amide hydrolysis

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

Professor David Liu and Brian Tse, Life Sciences 1a page 11 Acid Catalysis of Amide Hydrolysis

H ‡ N H Weaker electrophile: N O slower reaction, H ‡ O !" O O H higher ΔG H + H δ

H H ‡ Stronger electrophile: N N amide faster reaction, + H O O H ‡ acid O lower ΔG H H δ+ H δ+ O H • Acid can accelerate amide hydrolysis by protonating the amide oxygen, increasing its electrophilicity

During this attack by deprotonated water, an excess of electron density builds up on the oxygen atom of the amide group in the substrate. Without a nearby proton to neutralize this developing 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 other words, a neutral amide is not electrophilic enough to enable the nucleophile to efficiently form a bond. In the test tube, a chemist could add acid to the reaction to protonate the oxyanion, increasing the amide’s electrophilicity and stabilizing the transition state.

Professor David Liu and Brian Tse, Life Sciences 1a page 12 Acid Catalysis by HIV Protease

H N H H N ‡ N protonation O " of amide ! O O H H O O H O H δ− H H O H O O O H O H O O O O O O O

Asp 25’ Asp 25’ of HIV of HIV protease protease

• HIV protease precisely positions Asp 25’ to serve as an acid to protonate the substrate amide, increasing its electrophilicity and accelerating amide hydrolysis

Likewise, HIV protease uses acid catalysis to provide a proton to neutralize the forming oxyanion in the enzyme’s active site and therefore increase the substrate’s electrophilicity. Asp 25’, the other catalytic Asp residue, is protonated as the carboxylic acid in the active form of HIV protease. As the oxyanion on the substrate amide is being formed, the proton from Asp 25’ 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 water attacks the substrate’s amide group.

Professor David Liu and Brian Tse, Life Sciences 1a page 13 Enzymes Can Catalyze Reactions in Ways That Simple Acids and Bases Cannot

H H N N

O O H O H A O C H ID H H + H O O E S A - O O B H How can an acid and base :O simultaneously catalyze a single reaction? enzyme acid enzyme base

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

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

Professor David Liu and Brian Tse, Life Sciences 1a page 14 HIV Protease Active Site in Action

Ile 50’ attacking water

H substrate

Asp 25’ Asp 25

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

Professor David Liu and Brian Tse, Life Sciences 1a page 15 HIV Protease Catalysis Summary Proximity and orientation effects Tetrahedral H H intermediate N N ‡ H N O O O H H O H O O H H H H δ− O O O H δ− O O O O O O H O O O

Asp 25’ Asp 25 Acid Base catalysis catalysis

H H O N ‡ O H O H N H O O H O H N H − H H δ O H − O δ O O O H O O O O O O O

Base Acid catalysis catalysis

Integrating the above analysis forms the picture shown here for how HIV protease accelerates the rate of amide bond cleavage. Asp 25 serves as a catalytic base to deprotonate water. 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’s electrophilicity. After the formation of the tetrahedral intermediate), the catalytic Asp residues reverse their roles to complete the reaction. One of the hydroxyl groups in the tetrahedral intermediate is deprotonated by a catalytic aspartate (base catalysis), and the resulting anionic intermediate collapses. Instead of ejecting a hydroxide ion (which would represent exactly the reverse of the reaction thus far), the collapse of the tetrahedral intermediate causes the —NH group to break away. The departure of the —NH group (which in the absence of a catalyst would be negatively charged) is assisted by picking up a proton from a catalytic aspartic acid residue (acid catalysis). Therefore, a series of base-catalyzed and acid-catalyzed steps together with proximity and orientation effects collectively enable HIV protease to mediate the efficient hydrolysis of an otherwise very stable amide bond.

You may wonder how the tetrahedral intermediate “knows” to collapse to break the C-N bond instead of breaking a C-O bond. The answer is that the intermediate collapses in both ways— 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 mechanistic possibilities leading to a productive reaction step is a very common principle underlying the chemistry 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.

Professor David Liu and Brian Tse, Life Sciences 1a page 16 Lectures 15-16: The Molecular Basis of Enzyme Catalysis: HIV Protease 1. The function and structure of HIV protease a. Introduction to proteases b. Discovery of HIV protease c. Overview of the three-dimensional structure of HIV protease 2. Chemical reactions and the energies driving them a. Amide bond cleavage: the reaction catalyzed by HIV protease b. Thermodynamics of a reaction and free energy c. Kinetics of a reaction and ΔG‡ d. Reaction energy diagrams e. Transition states, intermediates, and how to draw them 3. How enzymes accelerate chemical reactions: the case of HIV protease a. Catalysts alter a reaction’s kinetics, but not its thermodynamics b. Chemical strategies behind enzyme catalysis i. Proximity and orientation effects ii. Nucleophilicity and electrophilicity iii. Acid and base catalysis 4. The molecular basis of substrate specificity a. Trypsin substrate specificity b. HIV protease substrate specificity

Professor David Liu and Brian Tse, Life Sciences 1a page 17 Protease Substrate Specificity

Scissile bond

S3 S1 S2’ S4’

O P3 O P O P ' O P ' H H 1 H 2 H 4 H N N N N N N N N N H O P O H H H 4 P2 O P1' O P3' O

S4 S2 S1’ S3’ Enzyme specificity pockets recognize the To N-terminal end specific amino acid To C-terminal end of substrate residues surrounding the of substrate bond to be hydrolyzed

4. Molecular basis of substrate specificity

For most enzymes, merely accelerating the rate of a chemical reaction that would otherwise proceed too slowly is not sufficient to be useful to a living system. Most enzymes must also be quite selective in the substrates that they convert to products to avoid interfering with other essential life processes. There is nothing about the catalytic strategy of HIV protease that would explain how it is able to exclusively cleave the correct amide bonds out of a vast number of possibilities. Instead, this specificity arises from many favorable interactions— mostly outside 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 examining trypsin, one of your digestive proteases. The major biological roles of trypsin in your body are to catalyze the hydrolysis of protein consumed as food. If trypsin catalyzed the hydrolysis of any peptide bond (i.e., if trypsin had no substrate specificity) then it would not only digest the proteins in your food but would also destroy other trypsin molecules as well as other important digestive enzymes. Fortunately, trypsin will only cleave proteins after exposed lysine or arginine residues. This simple substrate specificity helps trypsin avoid the haphazard hydrolysis 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 in the substrate that extend from the scissile bond toward the N-terminus of the substrate are designated P1, P2, P3, etc. Likewise, the amino acids that extend from the scissile bond toward the C-terminus of the substrate are called P1’, P2’, P3’, etc. The scissile bond is therefore the amide bond connecting the P1 and P1’ amino acids.

Professor David Liu and Brian Tse, Life Sciences 1a page 18 Trypsin: A Digestive Protease that Cleaves Substrates Containing Lys or Arg

substrate peptide enzyme trypsin substrate Asp189 enzyme Lys P1 S1 + –

scissile bond

trypsin active site residues

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

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 contains a negatively charged aspartic acid (Asp) 189 residue.

Professor David Liu and Brian Tse, Life Sciences 1a page 19 Basis of Trypsin Substrate Specificity

Asp 189 Asp 189 S1 S1 O O O O

H2N NH2 H3N NH Arg Lys H H P1 N P1 N N N H H O O • The presence of anionic Asp 189 in the S1 site causes a strong preference for P1 to be a cationic Lys or Arg

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

Professor David Liu and Brian Tse, Life Sciences 1a page 20 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 protease once again. There are roughly 1,500 peptide amide linkages in the Gag and Gag-Pro-Pol polyproteins, yet HIV protease cleaves only 10 of these amide bonds.

Understanding the basis of HIV protease’s substrate specificity is particularly important because inhibitors of HIV protease including those currently used to treat AIDS must be accepted by HIV protease and bind to the enzyme. Most enzyme inhibitors, including those of HIV protease, are molecules that mimic the shape and chemical properties of the substrate or transition state but cannot be converted into a product for chemical reasons. To be maximally effective, an enzyme inhibitor should ideally bind to the enzyme with an affinity that is greater than that of the natural substrate. Such an inhibitor can sequester the vast majority of the enzyme’s active sites in a cell, preventing the enzyme from efficiently processing its natural substrate.

Professor David Liu and Brian Tse, Life Sciences 1a page 21 The HIV Protease Active Site: A Closer Look

Note: structure is of an inactive Asn 25 mutant of HIV protease complexed with a substrate.

HIV protease substrate specificity

Professor David Liu and Brian Tse, Life Sciences 1a page 22 HIV Protease Substrate Selectivity

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

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

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

Professor David Liu and Brian Tse, Life Sciences 1a page 23 HIV Protease Specificity: P1 and P3

water S3 • HIV protease’s H H O structure can explain Gly 49 some aspects of its H H S1 NH O substrate speciﬁcity N N H H H N N • P1= large and HN H O hydrophobic; Arg P3 Leu P1 H complements O O H N H S1 = Gly 49 N N N N H H • P3 = polar oArsn 25’ O O O charged; S3 contains a bound water

Despite its ability to tolerate a variety of amino acids at most substrate positions, HIV protease makes a number of substrate interactions that are representative of the ways in which enzymes recognize their substrates. Shown here are some of the interactions between HIV protease and the P1 and P3 residues of one of its natural substrates. You can see that in the structure of the enzyme is well matched to bind the amino acids that commonly occur at the corresponding substrate position. For example, the P1 amino acid is usually large and hydrophobic, and the P1 side chain fits into a water-excluded cavity in the enzyme’s active site created by the absence of a side chain at Gly 49. Likewise, the preference of P3 for a polar or charged amino acid likely reflects the bound water molecule in the enzyme’s P3 binding pocket that 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 specificity are similar to those that enzymes make with transitions states to lower ΔG‡ and accelerate chemical reactions. If you take another look at the protease residues involved in proximity and orientation catalysis as well as base and acid catalysis you will find many of the same chemical themes. Conveying an intuitive understanding of protein-substrate and protein-transition state interactions is a major aspect of this course. Indeed, such an understanding recently enabled scientists to develop a major new class of HIV therapeutics that have had a major impact on extending the life of patients afflicted with AIDS. These small-molecule drugs, the HIV protease inhibitors, and the principles underlying their development are the subject of the next lectures in this course.

Professor David Liu and Brian Tse, Life Sciences 1a page 24 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 strategies to create a precise transition state-stabilizing active site environment • Enzymes use proximity and orientation effects to increase the concentration of substrates, increasing rates of reactions • Enzymes use acid and base catalysis to enhance the nucleophilicity or electrophilicity of reactants • Specificity arises from protein-substrate interactions

Professor David Liu and Brian Tse, Life Sciences 1a page 25