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Page 1 Proximity Effects on Reaction Rates 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.
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