Enzyme Mechanisms Andy Howard Biochemistry Lectures Spring 2019
Enzyme Mechanisms Andy Howard Biochemistry Lectures, Spring 2019 19 February 2019
Mechanisms matter Today’s inquiry is the mechanisms by which enzymes reduce activation energy barriers. n We’ll offer some examples of these mechanisms. n 02/19/2019 Mechanisms, Examples Page 2 of 73
Topics for today n Mechanisms n n Transition states Binding modes n n Proximity Transition-state stabilization Chemical modes n n Acid-base Covalent catalysis 02/19/2019 Mechanisms, concluded n n Induced fit Participating moieties Serine proteases Other mechanisms Mechanisms, Examples Page 3 of 73
How do enzymes reduce activation energies? n n We want to understand what is really happening chemically when an enzyme does its job. We’d also like to know how biochemists probe these systems. 02/19/2019 Mechanisms, Examples Page 4 of 73
Atomic-Level Mechanisms n n n We want to understand atomic-level events during an enzymatically catalyzed reaction. Sometimes we want to find a way to inhibit an enzyme in other cases we're looking for more fundamental knowledge, viz. the ways that biological organisms employ chemistry and how enzymes make that chemistry possible. 02/19/2019 Mechanisms, Examples Page 5 of 73
Overcoming the barrier Free Energy n Simple system: single high-energy transition state intermediate between reactants, products G‡ R P 02/19/2019 Reaction Coordinate Mechanisms, Examples Page 6 of 73
Activation energy & temperature n n It’s intuitively sensible that higher temperatures would make it easier to overcome an activation barrier Rate k(T) = Q 0 exp(- G‡/RT) G‡ = activation energy or Arrhenius energy This provides tool for measuring G‡ 02/19/2019 Mechanisms, Examples Svante Arrhenius Page 7 of 73
Determining G‡ n ca ta un ln k n ca lyz n ed n ta lyz ed Remember k(T) = Q 0 exp(- G‡/RT) ln k = ln. Q 0 - G‡/RT Measure reaction rate as function of temperature Plot ln k vs 1/T; slope will be - G‡/R 1/T, K-1 02/19/2019 Mechanisms, Examples Page 8 of 73
How enzymes alter G‡ n n Enzymes reduce G‡ by allowing the binding of the transition state into the active site Binding of the transition state needs to be tighter than the binding of either the reactants or the products. 02/19/2019 Mechanisms, Examples Page 9 of 73
G‡ and Entropy n n n Effect is partly entropic: When a substrate binds, it loses a lot of entropy. Thus the entropic disadvantage of (say) a bimolecular reaction is soaked up in the process of binding the first of the two substrates into the enzyme's active site. 02/19/2019 Mechanisms, Examples Page 10 of 73
Enthalpy and transition states n n Often an enthalpic component to the reduction in G‡ as well Ionic or hydrophobic interactions between the enzyme's active site residues and the components of the transition state make that transition state more stable. 02/19/2019 Mechanisms, Examples Page 11 of 73
Two ways to change G‡ n n Reactants bound by enzyme are properly positioned Get into transition-state geometry more readily E A B A+B 02/19/2019 n Transition state is stabilized E A B A+B A-B Mechanisms, Examples A-B Page 12 of 73
Binding and Chemistry n Catalysis via binding n n n Proximity effect Transition state stabilization Catalysis via chemistry n n Acid-base reactions Covalent catalysis 02/19/2019 Mechanisms, Examples Page 13 of 73
Binding modes: proximity n We describe enzymatic mechanisms in terms of the binding modes of the substrates (or, more properly, the transition-state species) to the enzyme. 02/19/2019 Mechanisms, Examples William Jencks Page 14 of 73
Proximity effect n One of these involves the proximity effect, in which two (or more) substrates are directed down potential-energy gradients to positions where they are close to one another. Thus the enzyme is able to defeat the entropic difficulty of bringing substrates together. 02/19/2019 Mechanisms, Examples Page 15 of 73
Binding modes: efficient transition-state binding n n Transition state fits even better (geometrically and electrostatically) in the active site than the substrate would. This improved fit lowers the energy of the transition-state system relative to the substrate. Best competitive inhibitors of an enzyme are those that resemble the transition state rather than the substrate or product. 02/19/2019 Mechanisms, Examples Page 16 of 73
Examples illustrating transition state stabilization n n Numerous enzymes act by providing stabilization of a transition state or an intermediate Giveaway is extremely effective competitive inhibitors that resemble the transition state that is being stabilized 02/19/2019 Mechanisms, Examples Page 17 of 73
Adenosine deaminase with transition-state analog n n Transition-state analog: Ki~10 -8 * substrate Km Wilson et al (1991) Science 252: 1278 02/19/2019 Mechanisms, Examples Page 18 of 73
ADA transition-state analog n 1, 6 hydrate of purine ribonucleoside binds with KI ~ 3*10 -13 M 02/19/2019 Mechanisms, Examples Page 19 of 73
Diffusion-controlled reactions n n Some enzymes are so efficient that the limiting factor in completion of the reaction is diffusion of the substrates into the active site: These are diffusion-controlled reactions. Ultra-high turnover rates: kcat ~ 108 s-1. We can describe kcat / Km as catalytic efficiency of an enzyme. A diffusion-controlled reaction will have a catalytic efficiency on the order of 108 M-1 s-1. 02/19/2019 Mechanisms, Examples Page 20 of 73
Induced fit (CF&M fig. 6. 4) n n n Refinement on original Emil Fischer lock-and-key notion: both the substrate (or transition-state) and the enzyme have flexibility Binding induces conformational changes 02/19/2019 Mechanisms, Examples Page 21 of 73
Ionic reactions (CF&M§ 7. 6) n n Define them as reactions that involve charged, or at least polar, intermediates Typically 2 reactants Electron rich (nucleophilic) reactant n Electron poor (electrophilic) reactant n 02/19/2019 Mechanisms, Examples Page 22 of 73
Describing nucleophilic substitutions n n Conventional to describe reaction as attack of nucleophile on electrophile Drawn with nucleophile donating electron(s) to electrophile 02/19/2019 Mechanisms, Examples Page 23 of 73
Attack on Acyl Group n n n Transfer of an acyl group Nucleophile Y attacks carbonyl carbon, forming tetrahedral intermediate X- is leaving group 02/19/2019 Mechanisms, Examples Page 24 of 73
Direct Displacement n n Attacking group adds to face of atom opposite to leaving group Transition state has five ligands; inherently less stable than schemes involving only four ligands on a carbon; but they still play out (Moran eqn. 6. 2) 02/19/2019 Mechanisms, Examples Page 25 of 73
Cleavage Reactions n Both electrons stay with one atom n n n Covalent bond produces carbanion: R 3—C—H R 3—C: - + H+ Covalent bond produces carbocation: R 3—C—H R 3—C+ + : H- One electron stays with each product n n n Both end up as radicals R 1 O—OR 2 R 1 O • + • OR 2 Radicals are reactive— some more than others 02/19/2019 Mechanisms, Examples Page 26 of 73
Covalent catalysis n n Refers to situations where a protein side -chain becomes directly involved in a (possibly unstable) covalent bond with one of the reactive species Example: serine proteases: see beginning of next lecture 02/19/2019 Mechanisms, Examples Page 27 of 73
Groups of amino acids n n n An enzyme generally has two or three absolutely critical side-chains that are directly involved in catalysis Specific examples discussed in section 6. 4 of your text: attend to those! Diads: Arg-arg, carboxylate-carboxylate, carboxylate-histidine 02/19/2019 Mechanisms, Examples Page 28 of 73
Metal ions in catalysis n n n Alkalai & alkaline earth: loosely bound, primarily playing structural roles Transition metals are often directly involved in catalysis, sometimes as Lewis acids Some transition metals can get involved in redox reactions too 02/19/2019 Mechanisms, Examples Page 29 of 73
Coenzymes n n n We’ll go into this in greater detail later Many are vitamins or are derived (via simple metabolic conversions) from vitamins Two categories: n n Cosubstrates (loosely bound, recycled) Prosthetic groups (tightly bound; restored to starting state in situ) 02/19/2019 Mechanisms, Examples Page 30 of 73
Temperature in enzymatic reactions n n Earlier discussion of Arrhenius: warmer = faster If the enzyme unfolds, it can’t catalyze the reaction at all So there’s an optimum temperature for any particular enzyme In homeothermic organisms the internal temperature is regulated anyway 02/19/2019 Mechanisms, Examples Page 31 of 73
p. H as an influence n n n Often there are two or three ionizable groups involved in a catalytic reaction Both (or all three) need to be in the correct protonation state in order for the reaction to proceed That sometimes imposes a specific optimal p. H for maximal enzyme activity 02/19/2019 Mechanisms, Examples Page 32 of 73
Oxidation-Reduction Reactions n n n Commonplace in biochemistry: EC 1 Oxidation is a loss of electrons Reduction is the gain of electrons 02/19/2019 Mechanisms, Examples Page 33 of 73
Analyzing redox reactions n In practice, often: n n n oxidation is decrease in # of C-H bonds; reduction is increase in # of C-H bonds Mnemonic: OIL RIG n n Oxidation is loss of electrons Reduction is gain of electrons 02/19/2019 Mechanisms, Examples Page 34 of 73
Redox, continued n n n Intermediate electron acceptors and donors are organic moieties or metals Ultimate electron acceptor in aerobic organisms is usually dioxygen (O 2) Anaerobic organisms usually employ other electron acceptors (Fe 3+, S, …) 02/19/2019 Mechanisms, Examples Page 35 of 73
Biological redox reactions I n n n Generally, but not always, these are 2 -electron transformations Often involve alcohols, aldehydes, ketones, carboxylic acids, C=C bonds: R 1 R 2 CH-OH + X R 1 R 2 C=O + XH 2 R 1 HC=O + X + OH- R 1 COO- + XH 2 X is usually NAD, NADP, FAD, FMN A few biological redox systems involve metal ions or Fe-S complexes 02/19/2019 Mechanisms, Examples Page 36 of 73
Biological Redox Reactions II n n FAD and FMN can operate one electron at a time, because they produce a quasi -stable free radical species in the middle Metal ion-mediated redox systems usually operate one electron at a time 02/19/2019 Mechanisms, Examples Page 37 of 73
Redox and energy n Usually reduced compounds are higherenergy than the corresponding oxidized compounds; therefore oxidation of reduced cofactors (NADH, NADPH, FADH 2) usually releases energy that can be harnessed for some other purpose 02/19/2019 Mechanisms, Examples Page 38 of 73
Induced fit Daniel Koshland n Cartoon from textbookofbacteriology. net 02/19/2019 Conformations of enzymes don't change enormously when they bind substrates, but they do change to some extent. An instance where the changes are fairly substantial is the binding of substrates to kinases. Mechanisms, Examples Page 39 of 73
Kinase reactions n n unwanted reaction ATP + H-O-H ⇒ ADP + Pi will compete with the desired reaction ATP + R-O-H ⇒ ADP + R-O-P Kinases minimize the likelihood of this unproductive activity by changing conformation upon binding substrate so that hydrolysis of ATP cannot occur until the binding happens. Illustrates the importance of the order in which things happen in enzyme function 02/19/2019 Mechanisms, Examples Page 40 of 73
Example of induced fit: hexokinase n n n Glucose + ATP Glucose-6 -P + ADP Risk: unproductive reaction with water Enzyme exists in open & closed forms Glucose induces conversion to closed form; water can’t do that Energy expended moving to closed form 02/19/2019 Mechanisms, Examples Page 41 of 73
Hexokinase conformational changes G&G Fig. 13. 28 02/19/2019 Mechanisms, Examples Page 42 of 73
Serine protease mechanism (CF&M § 6. 5, 7. 5) n n n Only detailed mechanism that we’ll ask you to memorize One of the first to be elucidated Well studied structurally Illustrates many other mechanisms Instance of convergent and divergent evolution 02/19/2019 Mechanisms, Examples Page 43 of 73
The reaction n Hydrolytic cleavage of peptide bond Enzyme usually works on esters too Found in eukaryotic digestive enzymes and in bacterial systems O NH 02/19/2019 CH R 1 NH C O Mechanisms, Examples C CH NH R-1 Page 44 of 73
How specific are proteases? n Widely-varying substrate specificities n n n Some proteases are highly specific for particular amino acids at position 1, 2, -1, . . . Others are more promiscuous Digestive proteases like chymotrypsin and trypsin are intermediate in specificity 02/19/2019 Mechanisms, Examples Page 45 of 73
Mechanism n n Active-site serine —OH … Without neighboring amino acids, it’s fairly unreactive becomes powerful nucleophile because OH proton lies near unprotonated N of His This N can abstract the hydrogen at near-neutral p. H Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate sidechain. 02/19/2019 Mechanisms, Examples Page 46 of 73
Catalytic triad n asp his The catalytic triad of asp, his, and ser is found in an approximately linear arrangement in all the serine proteases, all the way from nonspecific, secreted bacterial proteases to highly regulated and highly specific mammalian proteases. 02/19/2019 Mechanisms, Examples ser Page 47 of 73
Diagram of first three steps 02/19/2019 Mechanisms, Examples Page 48 of 73
Diagram of last four steps 02/19/2019 Diagrams courtesy University of. Page Virginia 49 of 73 Mechanisms, Examples
Chymotrypsin as example n n n Catalytic Ser is Ser 195 Asp is 102, His is 57 Note symmetry of mechanism: steps read similarly L R and R L 02/19/2019 Diagram courtesy of Anthony Serianni, University of Notre Dame Mechanisms, Examples Page 50 of 73
Oxyanion hole n n n When his-57 accepts proton from Ser-195: it creates an R—O- ion on Ser sidechain In reality the Ser O immediately becomes covalently bonded to substrate carbonyl carbon, moving negative charge to the carbonyl O. Oxyanion is on the substrate's oxygen 02/19/2019 Mechanisms, Examples Page 51 of 73
Stabilizing the oxyanion n Oxyanion stabilized by additional interaction in addition to the protonated his 57: main-chain NH group from gly 193 Hbonds to oxygen atom (or ion) from the substrate, further stabilizing the ion. 02/19/2019 Mechanisms, Examples Page 52 of 73
Oxyanion hole cartoon n Cartoon courtesy Henry Jakubowski, College of St. Benedict / St. John’s University 02/19/2019 Mechanisms, Examples Page 53 of 73
n n n Modes of catalysis in serine proteases Proximity effect: gathering of reactants in steps 1 and 4 Acid-base catalysis at histidine in steps 2 and 4 Covalent catalysis on serine hydroxymethyl group in steps 2 -5 Oxyanion hole stabilization in steps 3, 6 So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism 02/19/2019 Mechanisms, Examples Page 54 of 73
Specificity n n Active site catalytic triad is nearly invariant for eukaryotic serine proteases Remainder of cavity where reaction occurs varies significantly from protease to protease. 02/19/2019 Mechanisms, Examples Page 55 of 73
Chymotrypsin as example n n n In chymotrypsin hydrophobic pocket just upstream of the position where scissile bond sits This accommodates large hydrophobic side chain like that of phe, and doesn’t comfortably accommodate hydrophilic or small side chain. Thus specificity is conferred by the shape and electrostatic character of the site. 02/19/2019 Mechanisms, Examples Page 56 of 73
Chymotrypsin active site n n Comfortably accommodates aromatics at S 1 site Differs from other mammalian serine proteases in specificity 02/19/2019 Diagram courtesy University of Melbourne Mechanisms, Examples Page 57 of 73
Divergent evolution n Ancestral eukaryotic serine proteases presumably have differentiated into forms with different side-chain specificities Chymotrypsin is substantially conserved within eukaryotes, but is distinctly different from elastase Primary differences are in P 1 side chain pocket, but that isn’t inevitable 02/19/2019 Mechanisms, Examples Page 58 of 73
i. Clicker quiz, question 1 1. Why would the hexokinase reaction H 2 O + ATP ADP + Pi be considered nonproductive? n n n (a) Because it needlessly soaks up water (b) Because the enzyme undergoes a wasteful conformational change (c) Because the energy in the high-energy phosphate bond is unavailable for other purposes (d) Because ADP is poisonous (e) None of the above 02/19/2019 Mechanisms, Examples Page 59 of 73
i. Clicker Quiz question 2 2. What would bind tightest in the TIM active site? n (a) DHAP (substrate) n (b) D-glyceraldehyde 3 - phosphate (product) n (c) 2 -phosphoglycolate (Transition-state analog) n (d) They would all bind equally well n (e) None of them would bind at all. 02/19/2019 Mechanisms, Examples Page 60 of 73
i. Clicker quiz question 3 3. Consider the following serine proteases: n n n (1) porcine pancreatic elastase; (2) subtilisin from Bacillus subtilis; (3) porcine leukocyte elastase; (4) human pancreatic elastase. Which of these would be most similar to (1) ? (a) 2 (d) they’d all be equally similar (b) 3 (e) none would resemble #1. (c) 4 02/19/2019 Mechanisms, Examples Page 61 of 73
i. Clicker quiz question 4 4. Which of these words is correctly spelled? n (a) hydrophillic n (b) seperate n (c) tryptophane n (d) aniline n (e) none of the above 02/19/2019 Mechanisms, Examples Page 62 of 73
Convergent evolution n n Reappearance of ser-his-asp triad in unrelated settings Subtilisin: externals very different from mammalian serine proteases; triad same 02/19/2019 Mechanisms, Examples Page 63 of 73
Subtilisin mutagenesis n n Substitutions for any of the amino acids in the catalytic triad has disastrous effects on the catalytic activity, as measured by kcat. Km affected only slightly, since the structure of the binding pocket is not altered very much by conservative mutations. 02/19/2019 Mechanisms, Examples Page 64 of 73
Broken enzyme? n An interesting (and somewhat nonintuitive) result is that even these "broken" enzymes still catalyze the hydrolysis of some test substrates at much higher rates than buffer alone would provide. I would encourage you to think about why that might be true. 02/19/2019 Mechanisms, Examples Page 65 of 73
Cysteinyl proteases n n Ancestrally related to serine proteases? Cathepsins, caspases, papain 02/19/2019 Mechanisms, Examples Diagram courtesy of Mariusz Jaskolski, U. Poznan Page 66 of 73
Contrasting –SH with –OH n Contrasts: Cys —SH is more basic than ser —OH n Residue is less hydrophilic n S- is a weaker nucleophile than On 02/19/2019 Mechanisms, Examples Page 67 of 73
Papain active site 02/19/2019 Mechanisms, Examples Page 68 of 73
n n n Hen egg-white lysozyme Antibacterial protectant of growing chick embryo Hydrolyzes bacterial cell-wall peptidoglycans HEWL PDB 2 vb 1 “hydrogen atom of structural biology” n n n Commercially available in pure form Easy to crystallize and do structure work Available in multiple crystal forms n 02/19/2019 Mechanism is. Mechanisms, surprisingly complex Examples 0. 65Å 15 k. Da Page 69 of 73
Mechanism of lysozyme n n n Strain-induced destabilization of substrate makes the substrate look more like the transition state Long arguments about the nature of the intermediates Accepted answer: covalent intermediate between D 52 and glycosyl C 1 02/19/2019 Mechanisms, Examples Page 70 of 73
The controversy 02/19/2019 Mechanisms, Examples Page 71 of 73
Triosephosphate isomerase (TIM) n dihydroxyacetone phosphate glyceraldehyde-3 -phosphate DHAP 02/19/2019 Km=10µM kcat=4000 s-1 Glyc-3 -P kcat/Km=4*108 M-1 s-1 Mechanisms, Examples Page 72 of 73
TIM mechanism n n DHAP carbonyl H-bonds to neutral imidazole of his-95; proton moves from C 1 to carboxylate of glu 165 Enediolate intermediate (C—O- on C 2) Imidazolate (negative!) form of his 95 interacts with C 1—O-H; then glu 165 donates proton back to C 2 See Fort’s treatment http: //chemistry. umeche. maine. edu/ CHY 431/Enzyme 3. html) 02/19/2019 Mechanisms, Examples Leishmania TIM 56 k. Da dimer; monomer shown EC 5. 3. 1. 1 PDB 2 VXN, 0. 82Å Page 73 of 73
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