Enzymes How do Your Determine What is Going on at the Active Site

Enzymes How do your determine what is going on at the active site? We will discuss FOUR methods for study of the active site 1. 2. 3. 4. Enzyme kinetics pH studies Protein modification Structural studies 1 Enzymes 1) Use M-M Kinetics to determine the kinetic mechanism Steady-state kinetic analysis of bi-substrate reactions •Is it sequential random or ping-pong bi bi? •We cannot easily distinguish sequential ordered from ping-pong. Recall: In enzyme inhibition, if S MUST bind before I, you get parallel lines, and if I can bind to both forms of the enzyme (E and ES) you get x-axis intersecting lines. Its that same principle for bi-substrate enzymes: if A MUST bind before B, you get parallel lines, and if B can bind to both forms E and EA of the enzyme, you get x-axis intersecting lines. Ping-pong bi bi Sequential ordered bi bi A AB P Q Sequential random bi bi B Enzymes In these double-reciprocal plots, the concentration of A is varied while the concentration of B is held constant (at less than saturating concentrations). This is repeated for several values of [B], generating several separate lines. Sequential random bi bi B B A Lineweaver-Burk: lines are parallel A Lineweaver-Burk: lines intersect Ping-pong bi bi OR Sequential ordered bi bi In both cases, A must bind before B 2 Enzymes 2) Use M-M Kinetics to determine if there is acid-base catalysis Lets suppose you assay enzyme 1 at varying pH values. And, you get this: Then on enzyme 2, you get this: BH ← B: AH → A– !0 !0 3 4 5 6 7 pH 8 9 10 11 5 What is the difference? What does this mean in each case? What other information can you get? 5.5 6 6.5 7 pH 7.5 8 8.5 9 Enzymes 2) Use M-M Kinetics to determine if there is acid-base catalysis Lets take enzyme 2 and determine the values of Vmax and Km at varying pH values. What are you treating protons as? Now, plot the Vmax and Vmax/Km versus [H+] (i.e., pH) Do the pKa (Ka≈Ki) values give your any clue as to what residues are functioning as acid/base catalysts at the active site? If pKE1 = 4.5 ?Glu must be de-protonated for binding If pKES2 = 9.5 ?Lys must be protonated for catalysis 3 Enzymes 3) Use protein modification to determine what residues might be AT the active site If you react your enzyme with chemical reagents that are specific to certain amino acid residues, and these residues are at the active site, you might abolish activity. Certain controls are usually required: make sure that reagent doesn’t just denature the enzyme test to see if substrates or competitive inhibitors will protect should measure stoichiometry of reaction Common Reagents for the Modification of Proteins Reagent Residue 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent) N-bromosuccinimide Phenylisothiocyante (Edman's Reagent) Iodoacetic acid N-ethylmaleimide (NEM) 5,5'-Dithiobis(2-nitrobenzoic acid) (Ellman's Reagent - DTNB) Diethylpyrocarbonate (DEPC) Imidates 2,4,6-trinitrobenzenesulfonic acid (TNBS) Detection Tryptophan 410 nm Tryptophan Amino-terminal Cysteine Cysteine Cysteine 260/280 nm Release of a PTH-amino acid Carboxylmethyl derivatives Derivatives of NEM 412 nm Histidine Lysine Lysine 240 nm Derivatives of imidates 420 nm How can you use this idea and identify WHICH of the many His, Cys, Lys, etc. might be the one at the active site? Enzymes 3) Use protein modification to determine what residues might be AT the active site Use a “Trojan Horse” This combines the specificity of binding at the active site with the reactivity of the reagent for certain residues Examples: This will specifically Kill chymotrypsin General term: Affinity Labeling You can then perform protein sequencing studies to find which residue is modified 4 Enzymes 4) Use structural studies to SEE the active site X-ray crystallography can often reveal a cleft, which is usually that active site. Can look in the cleft for metal ions, coenzymes, acid/base groups, and/or nucleophiles Examples: You can then test which residues you actually see at the active site by protein modification, pH studies, or site-directed mutagenesis. Enzymes TRANSITION-STATE THEORY: Energetics SUMMARY SO FAR: We have described enzymes in general terms such as: catalytic cycle binding, even stereo-specific binding catalysis, turnover number & proficiency nomenclature transition state theory catalytic strategies (what to do) mechanistic strategies (how to do) enyzme kinetics and inhibition Deciphering the ACTIVE SITE Can we Quantify the energy needed to get the kind of rate enhancements enzymes enjoy? 5 10/20/23 Enzymes What enzyme energetics are involved in lowering the activation energy? Now that we can discover what the “geography” of the active site might be, lets discuss what has to happen there… Recall Transition State Theory and the 4 Catalytic Strategies: Catalytic Strategies DDG‡ – Position Effects: bind substrates where they need to be for reaction (rather than depending on random collisions) – Polarization of bonds: make substrates more reactive by polarizing bonds (make better nucleophiles, – (Electrostatics) electrophile, or leaving groups) DDG‡ = DG‡cat – DG‡uncat There is always a difference in activation energies that yield a negative DDG‡. This is the amount of energy that must be supplied somehow to the reaction by the enzyme – Strain of bonds: bind substrates in such a way that they ”look” like products (put strain on bonds that are to be (Geometry) broken (sessile)) – De-solvation: assist in removal of water shell around substrates or adding to products upon release (S & P are usually in direct contact with residues at the active site (no water)) Enzymes Enzymes organize reactive groups into close proximity and proper orientation. •Whatever way they do this, they have to have a negative DDG‡ DDG‡ = DDH‡ – TDDS‡ •This can be from strong polarizing bonds in ES & ES‡ • Catalyzed bimolecular and unimolecular reactions •This can come MUST use binding energy from differences in from somewhere to, not only energy of the solvated S and ES pay the entropic cost of complex organizing the reactants into a fairly rigid ES complex Catalyzed – Uncatalyzed staged to achieve the (more bonds; (fewer bonds; lower energy higher energy transition state, but also to less enthalpy) more enthalpy) get enough energy to lower DDH‡ Value is: – – the activation energy. This DDH‡ MUST be much more – • Catalyzed bimolecular and unimolecular reactions have to position reactants and/or strain them to reach the transition state in the active site than – TDDS‡ is Catalyzed – Uncatalyzed (less S) (more S) DDS‡ Value is: – TDDS‡ value is: – + + 6 Enzymes An easy way to express this is simply to say that: Enzymes bind transition states best. • This idea was proposed by Linus Pauling in 1946. – Enzyme active sites are complimentary to the transition state of the reaction. – Enzymes bind transition states better than substrates. – Stronger/additional interactions with the transition state as compared with the ground state lower the activation barrier. As shown on last slide, this is largely ΔΔH‡ effect What is an example of using binding energy to increase rates? Enzymes The rate of anhydride formation (condensation of two acids) from an esters and carboxylates shows a strong dependence on proximity of two reactive groups, “paid” by covalent bonds (work by Thomas C. Bruice’s group). Relative Rate 1 6.0 x 102 1.2 x 103 1.0 x 105 6.0 x 106 For an enzyme, how much binding energy is needed to increase the rate by 106? 3.0 x 107 7 Enzymes • Enzymes increase reaction rates (!0) by decreasing ΔG‡. !0 = kcat [E]T ⎠ ⎝ ⎠ cat = ⎝ The change in rate constant from the uncatalyzed: Dk ⎝ ⎠ –DDG‡ kBT RT e h ⎝ ⎠ kB = Boltzmann’s constant (J/°K) h = Plank’s constant (J•sec) T = Temperature (°K) R = Gas constant (J•°K-1•mol-1) TWO points about this equation: 1) the relationship between DDG‡ and rate is negative; the higher the negative value, the larger the rate 2) the relationship between DDG‡ and rate is exponential; a small change in energy, a large change in rate DDG‡ (kcal/mole) Dkcat (s-1) –1.4 101 –2.8 102 –5.6 104 –8.0 106 Enzymes Binding Energy is the free energy released upon interaction of the enzyme and substrate. Binding Energy need NOT be just in the interactions directly with the substrate; it could be that Binding Energy (bonds) is gained from the entire protein (enzyme dynamics) in the ES complex. It has been proposed that the ES complex is a high-energy state and the ES‡ complex is a low-energy state. Upon S binding, the ES complex is sort of a “wound-up” protein, and Binding Energy helps force the ES à ES‡ reaction, i.e., ES‡ is more easily achieved as the whole protein finds a lower energy state. In other words, it seems clear now that Binding Energy is greatest when the enzyme interacts as it approaches the transition state, thus facilitating the formation of the transition state. EXAMPLE: The racemization of proline proceeds through a transition state in which the α-carbon is trigonal. This reaction is catalyzed by Proline Racemase. 8 Enzymes EXAMPLE: The racemization of proline proceeds through a transition state in which the α-carbon is trigonal. This reaction is catalyzed by Proline Racemase. sp2carbanion 4 5 5 2 1 +H 4 3 – HA-E-B: 5 2 1 +H 2 4 3 – HA-E-BH 2 1 +H 2 3 – 2 –A- -BH E Other substrates/inhibitors that have a trigonal geometry (sp2) might look more like the transition state, so called transition-state analogs. These might bind better than S. Relative Kd values: L-Proline Tetrahydrofuran2-carboxylate D-1-Pyrroline2-carboxylate Pyrrole-2carboxylate 1 2 0.006 0.006 A potent inhibitor of proline racemase. Enzymes EXAMPLE: HIV protease This protease is important in the processing of the viral proteins and is encoded by the HIV genome. It was the first successful target for treatment of HIV. Saved millions of lives to date. This is a protease that uses an Asp at the active site (Asp-protease or acid protease), but it has a specificity for cleavage at a Tyr/Phe – Pro bond (among others). sp3tetrahedral Kd = 0.4 nM What are all these other substituents doing? Kd = 0.015 nM In drug development, issues of Bioavailability are paramount: survival in the gut, absorption, half-life, membrane permeability, off-target minimization 9