Andy Howard Introductory Biochemistry, Spring 2008 19 February 2008
Enzyme Mechanisms Andy Howard Introductory Biochemistry, Spring February 2008 Biochemistry: Enzyme Mechanisms 19 Feb 2008 How do enzymes reduce activation energies?
We want to understand what is really happening chemically when an enzyme does its job. Wed also like to know how biochemists probe these systems. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Mechanism Topics Terminology Binding Modes of Catalysis
Transition States Enzyme chemistry Diffusion-controlled Reactions Binding Modes of Catalysis Induced-fit Tight Binding of Ionic Intermediates Serine proteases Biochemistry: Enzyme Mechanisms 19 Feb 2008 Functional Note Henceforth we wont be providing as much textbook-independent stuff Well rely more heavily on Hortons own treatments If you dont understand something in Horton, ask me or one of the other IIT biochemists Well provide clear syllabi for exams Biochemistry: Enzyme Mechanisms 19 Feb 2008 Atomic-Level Mechanisms
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. Biochemistry: Enzyme Mechanisms 19 Feb 2008 How we study mechanisms
There are a variety of experimental tools available for understanding mechanisms, including isotopic labeling of substrates, structural methods, and spectroscopic kinetic techniques. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Ionic reactions Define them as reactions that involve charged, or at least polar, intermediates Typically 2 reactants Electron rich (nucleophilic) reactant Electron poor (electrophilic) reactant Conventional to describe reaction as attack of nucleophile on electrophile Drawn with nucleophile donating electron(s) to electrophile Biochemistry: Enzyme Mechanisms 19 Feb 2008 Attack on Acyl Group Transfer of an acyl group: scheme 6.1
Nucleophile Y attacks carbonyl carbon, forming tetrahedral intermediate X- is leaving group Biochemistry: Enzyme Mechanisms 19 Feb 2008 Direct Displacement Attacking group adds to face of atom opposite to leaving group (scheme 6.2) Transition state has five ligands; inherently less stable than scheme 6.1 Biochemistry: Enzyme Mechanisms 19 Feb 2008 Cleavage Reactions Both electrons stay with one atom
Covalent bond produces carbanion: R3CH R3C:- + H+ Covalent bond produces carbocation: R3CH R3C+ + :H- One electron stays with each product Both end up as radicals R1OOR2 R1O + OR2 Radicals are highly reactive some more than others Biochemistry: Enzyme Mechanisms 19 Feb 2008 Oxidation-Reduction Reactions
Commonplace in biochemistry: EC 1 Oxidation is a loss of electrons Reduction is the gain of electrons In practice, often: oxidation is decrease in # of C-H bonds; reduction is increase in # of C-H bonds Intermediate electron acceptors and donors are organic moieties or metals Ultimate electron acceptor in aerobic organisms is usually dioxygen (O2) Biochemistry: Enzyme Mechanisms 19 Feb 2008 Biological redox reactions
Generally 2-electron transformations Often involve alcohols, aldehydes, ketones, carboxylic acids, C=C bonds: R1R2CH-OH + X R1R2C=O + XH2 R1HC=O + X + OH- R1COO- + XH2 X is usually NAD, NADP, FAD, FMN A few biological redox systems involve metal ions or Fe-S complexes Usually reduced compounds are higher-energy than the corresponding oxidized compounds Biochemistry: Enzyme Mechanisms 19 Feb 2008 Overcoming the barrier
Simple system: single high-energy transition state intermediate between reactants, products Free Energy G R P Reaction Coordinate Biochemistry: Enzyme Mechanisms 19 Feb 2008 Intermediates Often there is a quasi-stable intermediate state midway between reactants & products; transition states on either side T2 T1 Free Energy I R P Reaction Coordinate Biochemistry: Enzyme Mechanisms 19 Feb 2008 Activation energy & temperature
Its intuitively sensible that higher temperatures would make it easier to overcome an activation barrier Rate k(T) = Q0exp(-G/RT) G = activation energy or Arrhenius energy This provides tool for measuring G Svante Arrhenius Biochemistry: Enzyme Mechanisms 19 Feb 2008 Determining G Remember k(T) = Q0exp(-G/RT) ln k = lnQ0 - G/RT
Measure reaction rate as function of temperature Plot ln k vs 1/T; slope will be -G/R catalyzed ln k uncatalyzed 1/T, K-1 Biochemistry: Enzyme Mechanisms 19 Feb 2008 How enzymes alter G Enzymes reduce DG 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. Biochemistry: Enzyme Mechanisms 19 Feb 2008 DG and Entropy 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. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Enthalpy and transition states
Often an enthalpic component to the reduction in DG 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. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Two ways to change G Reactants bound by enzyme are properly positioned Get into transition-state geometry more readily Transition state is stabilized A B A B E E A+B A+B A-B A-B Biochemistry: Enzyme Mechanisms 19 Feb 2008 Reactive sidechains in a.a.s
Group Functions Asp COO- -1 Cation binding, proton transfer Glu Same as above His Imidazole ~0 Proton transfer Cys CH2SH Covalent binding of acyl gps Tyr Phenol H-bonding to ligands Lys NH3+ +1 Anion binding, proton transfer Arg guanadinium Anion binding Ser CH2OH See cys Biochemistry: Enzyme Mechanisms 19 Feb 2008 Generalizations about active-site amino acids
Typical enzyme has 2-6 key catalytic residues His, asp, arg, glu, lys account for 64% Remember: pKa values in proteins sometimes different from those of isolated aas Frequency overall Frequency in catalysis Biochemistry: Enzyme Mechanisms 19 Feb 2008 Cleavages by base : : Simple cleavage: XH + :B X:- + HB+
This works if X=N,O; sometimes C Removal of proton from H2O to cleave C-X: O O O- COH + HN CN CN : : HO :B O HB+ H H :B Biochemistry: Enzyme Mechanisms 19 Feb 2008 Cleavage by acid Covalent bond may break more easily if one of its atoms is protonated Formation of unstable intermediate, R-OH2+, accelerates the reaction Example:R+ + OH- ROH ROH2+ R+ + H2O (Slow) (Fast) Biochemistry: Enzyme Mechanisms 19 Feb 2008 Covalent catalysis Reactive side-chain can be a nucleophile or an electrophile, but nucleophile is more common AX + E XE + A XE + B BX + E Example: sucrose phosphorylase Net reaction: Sucrose + Pi Glucose 1-P + fructose Fructose=A, Glucose=X, Phosphate=B Biochemistry: Enzyme Mechanisms 19 Feb 2008 Rates often depend on pH
If an amino acid that is necessary to the mechanism changes protonation state at a particular pH, then the reaction may be allowed or disallowed depending on pH Two ionizable residues means there may be a narrow pH optimum for catalysis Biochemistry: Enzyme Mechanisms 19 Feb 2008 Papain as an example Biochemistry: Enzyme Mechanisms 19 Feb 2008 Diffusion-controlled reactions
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 ~ 109 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-1s-1. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Triosephosphate isomerase (TIM)
dihydroxyacetone phosphate glyceraldehyde-3-phosphate Glyc-3-P DHAP Km=10M kcat=4000s-1 kcat/Km=4*108M-1s-1 Biochemistry: Enzyme Mechanisms 19 Feb 2008 TIM mechanism DHAP carbonyl H-bonds to neutral imidazole of his-95; proton moves from C1 to carboxylate of glu165 Enediolate intermediate (CO- on C2) Imidazolate (negative!) form of his95 interacts with C1O-H) glu165 donates proton back to C2 See Forts treatment or fig. 6.7. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Examining enzyme mechanisms will help us understand catalysis
Examining general principles of catalytic activity and looking at specific cases will facilitate our appreciation of all enzymes Biochemistry: Enzyme Mechanisms 19 Feb 2008 Binding modes: proximity
We describe enzymatic mechanisms in terms of the binding modes of the substrates (or, more properly, the transition-state species) to the enzyme. 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. William Jencks Biochemistry: Enzyme Mechanisms 19 Feb 2008 Binding modes: efficient transition-state binding
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. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Adenosine deaminase with transition-state analog
Transition-state analog: Ki~10-8 * substrate Km Wilson et al (1991) Science 252: 1278 Biochemistry: Enzyme Mechanisms 19 Feb 2008 Induced fit Refinement on original Emil Fischer lock-and-key notion:
both the substrate (or transition-state) and the enzyme have flexibility Binding induces conformational changes Biochemistry: Enzyme Mechanisms 19 Feb 2008 Example: hexokinase Glucose + ATP Glucose-6-P + ADP
Risk: unproductive reaction with water Enzyme exists in open & closed forms Glucose induces conversion to closed form; water cant do that Energy expended moving to closed form Biochemistry: Enzyme Mechanisms 19 Feb 2008 Hexokinase structure Diagram courtesy E. Marcotte, UT Austin
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Tight binding of ionic intermediates
Quasi-stable ionic species strongly bound by ion-pair and H-bond interactions Similar to notion that transition states are the most tightly bound species, but these are more stable Biochemistry: Enzyme Mechanisms 19 Feb 2008 Serine protease mechanism
Only detailed mechanism that well ask you to memorize One of the first to be elucidated Well studied structurally Illustrates many other mechanisms Instance of convergent and divergent evolution Biochemistry: Enzyme Mechanisms 19 Feb 2008 The reaction Hydrolytic cleavage of peptide bond
Enzyme usually works on esters too Found in eukaryotic digestive enzymes and in bacterial systems Widely-varying substrate specificities Some proteases are highly specific for particular aas at position 1, 2, -1, . . . Others are more promiscuous CH NH C NH C NH R1 CH O Biochemistry: Enzyme Mechanisms R-1 19 Feb 2008 Mechanism Active-site serine OH Without neighboring amino acids, its fairly non-reactive becomes powerful nucleophile because OH proton lies near unprotonated N of His This N can abstract the hydrogen at near-neutral pH Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate side-chain. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Catalytic triad The catalytic triad of asp, his, and ser is found in an approximately linear arrangement in all the serine proteases, all the way from non-specific, secreted bacterial proteases to highly regulated and highly specific mammalian proteases. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Diagram of first three steps
Biochemistry: Enzyme Mechanisms 19 Feb 2008 Diagram of last four steps
Diagrams courtesy University of Virginia Biochemistry: Enzyme Mechanisms 19 Feb 2008 Chymotrypsin as example
Catalytic Ser is Ser195 Asp is 102, His is 57 Note symmetry of mechanism: steps read similarly L R and R L Diagram courtesy of Anthony Serianni, University of Notre Dame Biochemistry: Enzyme Mechanisms 19 Feb 2008 Oxyanion hole When his-57 accepts proton from Ser-195: it creates an RO- ion on Ser sidechain In reality the Ser O immediately becomes covalently bonded to substrate carbonyl carbon, moving - charge to the carbonyl O. Oxyanion is on the substrate's oxygen Oxyanion stabilized by additional interaction in addition to the protonated his 57: main-chain NH group from gly 193 H-bonds to oxygen atom (or ion) from the substrate, further stabilizing the ion. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Oxyanion hole cartoon Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.Johns University Biochemistry: Enzyme Mechanisms 19 Feb 2008 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 So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism Biochemistry: Enzyme Mechanisms 19 Feb 2008 Specificity Active site catalytic triad is nearly invariant for eukaryotic serine proteases Remainder of cavity where reaction occurs varies significantly from protease to protease. In chymotrypsin hydrophobic pocket just upstream of the position where scissile bond sits This accommodates large hydrophobic side chain like that of phe, and doesnt comfortably accommodate hydrophilic or small side chain. Thus specificity is conferred by the shape and electrostatic character of the site. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Chymotrypsin active site
Comfortably accommodates aromatics at S1 site Differs from other mammalian serine proteases in specificity Diagram courtesy School of Crystallography, Birkbeck College Biochemistry: Enzyme Mechanisms 19 Feb 2008 Divergent evolution 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 Biochemistry: Enzyme Mechanisms 19 Feb 2008 iClicker quiz! Why would the nonproductive hexokinase reaction H2O + ATP -> ADP + Pi be considered nonproductive? (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 Biochemistry: Enzyme Mechanisms 19 Feb 2008 iClicker quiz, question 2: Why are proteases often synthesized as zymogens?
(a) Because the transcriptional machinery cannot function otherwise (b) To prevent the enzyme from cleaving peptide bonds outside of its intended realm (c) To exert control over the proteolytic reaction (d) None of the above Biochemistry: Enzyme Mechanisms 19 Feb 2008 Question 3: what would bind tightest in the TIM active site?
(a) DHAP (substrate) (b) D-glyceraldehyde (product) (c) 2-phosphoglycolate (Transition-state analog) (d) They would all bind equally well Biochemistry: Enzyme Mechanisms 19 Feb 2008 Convergent evolution Reappearance of ser-his-asp triad in unrelated settings Subtilisin: externals very different from mammalian serine proteases; triad same Biochemistry: Enzyme Mechanisms 19 Feb 2008 Subtilisin mutagenesis
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. An interesting (and somewhat non-intuitive) 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. Biochemistry: Enzyme Mechanisms 19 Feb 2008 Cysteinyl proteases Ancestrally related to ser proteases?
Cathepsins, caspases, papain Contrasts: Cys SH is more basic than ser OH Residue is less hydrophilic S- is a weaker nucleophile than O- Diagram courtesy of Mariusz Jaskolski, U. Poznan Biochemistry: Enzyme Mechanisms 19 Feb 2008 Papain active site Diagram courtesy Martin Harrison, Manchester University Biochemistry: Enzyme Mechanisms 19 Feb 2008 Comments on the midterm
One remote student still hasnt taken it One local student still hasnt taken it I have graded A-N plus a couple of Ps You may pick up the exams beginning Thursday It was long, but many students finished I dont do grade cutoffs, but if I did, I would guess that theyre at 150 and 110. Rebecca Howard, Ph.D. Biochemistry: Enzyme Mechanisms 19 Feb 2008