SF Chemical Kinetics

Transcription

1 SF Chemical Kinetics Lecture 4. Catalysis (II): Enzyme biocatalysis and biosensors. What are Biosensors? Biosensors combine the exquisite selectivity of biology with the processing power of modern microelectronics and optoelectronics to offer powerful new analytical tools with major applications in medicine, environmental diagnostics and the food and processing industries. Biosensors consist of biorecognition systems, typically enzymes or binding proteins, such as antibodies, immobilised onto the surface of physicochemical transducers. In addition to enzymes and antibodies, the bio-recognition systems can also include nucleic acids, bacteria and single cell organisms and even whole tissues of higher organisms. Specific interactions between the target analyte and the complementary biorecognition layer produces a physicochemical change which is detected and may be measured by the transducer. The transducer can tae many forms depending upon the parameters being measured - electrochemical, optical, mass and thermal changes are the most common. 1

2 Biosensor:formal definition Chemical/biological receptor microstructure (where there is a specific molecular interaction between receptor and analyte species) coupled to an electronic transducer which converts chemical/biochemical activity into electrical signals which can be amplified, stored, displayed and manipulated. A biosensor is a device that recognizes an analyte in an appropriate sample and interprets its concentration as an electrical signal via a suitable combination of a biological recognition system and a suitable transducer.

3 Chemical/Biological Sensor Configuration. chemical interaction translated into a useful signal Receptor Electronics Substrate (analyte) Interferent specific molecular recognition between substrate and receptor site Transducer data transformed and processed into useful format Foundations of a viable sensor technology Chemical/biological sensor technology involves the interplay of many fundamental scientific and engineering disciplines. Chemistry (Physical, analytical, synthetic) Materials science Physics (solid state, optical) Biology (biochemistry: enzyme/ substrate, antibody/antigen etc) Engineering (electronics, ICT, microfabrication etc.). 3

4 Electrochemistry : Recap of basic ideas. Electrochemistry defines a branch of chemistry which deals with the chemical action of electricity and the production of electricity by chemical reactions. Key event is heterogeneous electron transfer (HET) at electrode/solution interface. HET is driven via application of external electrical potential Current flow (proportional to HET reaction rate) across electrode/solution interface reflects chemistry occurring there. Analytical aspect: Any redox active species can in principle be detected since current directly proportional to analyte concentration (provided diffusion control pertains). Interfacial electron transfer at electrode/solution interfaces: oxidation and reduction processes. Electron sin electrode (Anode). Electron source electrode (Cathode). P A ne - Q ne - B xidation (de-electronation). P Reductant (electron donor) Q xidant (product) Reduction (electronation). A oxidant (electron acceptor). B reductant (product). In principle any species which can be oxidised or reduced can be detected amperometrically. 4

5 Electrochemical sensors. Potentiometric devices. Local equilibrium established at the sensor/environment interface. Electrode or membrane potential measured. Equilibrium potential proportional to the logarithm of the analyte concentration via the Nernst equation. Amperometric devices. The electrode potential is used to drive an interfacial redox reaction and the current resulting from that reaction is measured. The current flowing is directly proportional to the analyte concentration. i Amperometric measurement made in mass transfer limited region. c 1.0 Current response ψ i / i D 0.5 Amperometric measurement: Apply fixed potential in mass transfer limited region, and measure current flow. Repeat for various analyte concentrations. Current directly proportional to analyte concentration ξ F(E-E 0 )/RT Applied potential (driving force) 5

6 Amperometric Glucose Sensors A. Heller, B. Feldman, Electrochemical glucose sensors and their applications in diabetes management. Chem. Rev. 008, in press. J. Wang, Electrochemical glucose biosensors. Chem. Rev., 108 (008) J. Wang, Glucose biosensors: 40 years of advances and challenges. Electroanalysis, 13 (001) J. Wang, In vivo glucose monitoring: towards sense and act feedbac loop individualized medical systems. Talanta, 75 (008) Amperometric enzyme biosensors. Enzymes are very specific biological catalysts. They interact with substrates via the Michaelis/Menten mechanism. If enzymes can be incorporated and immobilized within a matrix located next to an electrode surface, then it is possible to combine the specificity of enzyme catalysis with the many advantages of amperometric detection. Some questions need addressing before this useful synergy can be achieved. How can enzymes be immobilized in a region next to an electrode surface? How can the enzyme be made to communicate with the underlying support electrode? How can we maintain the catalytic integrity of the immobilized enzyme? How can we describe the mechanism and quantify the inetics underlining the operation of an amperometric enzyme electrode? We will focus attention on redox enzymes, and in particular, glucose oxidase. 6

7 Kinetics of enzyme reactions. Enzymes are very specific biological catalysts. A catalyst is a substance that increases the rate of a reaction without itself being consumed by the process. A catalyst lowers the Gibbs energy of activation ΔG by providing a different mechanistic pathway by which the reaction may proceed. This alternative mechanistic path enhances the rate of both the forward and reverse directions of the reaction. The catalyst forms an intermediate with the reactants in the initial step of the reaction ( a binding reaction), and is released during the product forming step. Regardless of the mechanism and reaction energetics a catalyst does not effect ΔH or ΔG of the reactants and products. Hence catalysts increase the rate of approach to equilibrium, but cannot alter the value of the thermodynamic equilibrium constant. energy thermodynamics unchanged E A lowered ΔE A reactants ΔH reaction coordinate catalyst absent catalyst present products A reactant molecule acted upon by an enzyme is termed a substrate. The region of the enzyme where the substrate reacts is called the active site. Enzyme specificity depends on the geometry of the active site and the spatial constraints imposed on this region by the overall structure of the enzyme molecule. Space filling models of the two conformations of the enzyme hexoinase. (a) the active site is not occupied. There is a cleft in the protein structure that allows the substrate molecule glucose to access the active site. (b) the active site is occupied. The protein has closed around the substrate. glucose H CH H H H H H H H H + ATP hexoinase - CH P 3 H H H H H + ADP + H + H H H H hexoinase glucose glucose glucose 6- phosphate Enzyme loc/ey mechanism : natural molecular recognition. 7

8 Chymotrypsin : A digestive enzyme. substrate active site Natural molecular recognition in action. binding pocet Redox enzymes. Redox enzyme contains tightly bound redox active prosthetic group (e.g. flavin, haem, quinone) that remains bound to the protein throughout redox cycle. Prosthetic group non amino acid component of conjugated protein. Redox enzymes exist in both oxidised and reduced forms. Redox enzymes can be subclassified in terms of the redox centres present in the enzyme. Flavoproteins are most often studied. They consist of ca. 80 different enzymes containing either Flavin adenine dinucleotide (FAD) Flavin mononucleotide (FMN) at the active site. The flavin unit is strongly associated with the protein structure and is sometimes covalently bound to the amino acid residues in enzyme. 8

9 Glucose oxidase b-d-glucose: oxygen 1-oxidoreductase EC ) : Gx. Dimeric structure of glucose oxidase Gx. Gx is a dimeric protein with MW 160 Da. Contains one tightly bound flavin adenine dinucleotide FAD unit per monomer as cofactor. FAD is not covalently bound and can be released from the holo protein following denaturation. FAD exhibits redox activity. Gox exhibits a very high degree of specificity for β-d-glucose. monomeric unit redox active region protein sheath FAD active site Structure of FAD redox site in Gx. 9

10 Redox chemistry of flavin Groups. Flavin redox system involves the transfer of two electrons, and also involves associated protonation/deprotonation equilibria. Hence the redox behaviour will depend on the ph of the environment. Redox chemistry involves quinone, semiquinone and hydroquinone states. The latter may be subject to various degrees of protonation. Redox properties of free flavin are well understood. FAD/FADH, ph 7, E 0-568mV vs SCE Results obtained for the free flavin not directly applicable to the situation of a flavin group located within the protein envelope of an enzyme because the environment is different. Hence local ph can differ from bul ph of solution and also local environment is enzyme specific. Redox potential of flavin group within a protein environment at ph7 can vary over the range -730 to + 50 mv (SCE). Environment at active site of redox enzyme has profound effect on redox chemistry of FAD centres. Redox potential shift occurs and relative stability of flavin, semiflavin and reduced flavin forms are altered. xidised form Reduced form Mechanism of enzyme action. 10

11 Classification of enzymes. 11

12 Time course of enzyme catalyzed reaction. Assume that [S] >> [E] Note logarithmic timescale. Note linear timescale. Enzyme catalysed reaction: Variation of reaction rate with substrate (reactant) concentration ΨR / c e Unsaturated enzyme inetics Saturated enzyme inetics uc/k M Rate variation adopts shape of rectangular hyperbola. This is an example of a complex rate equation, where the reaction rate varies with reactant concentration in a non linear way. 1

13 Kinetics of enzyme reactions : Simple Michaelis-Menten Mechanism. 1 E + S ES E + P -1 x [ ES] c [ S] e [ E] dx dt ss ec x x 1 ss ss 0 e e + x ( ) e x c x x 1 ss ss ss 0 R ce 1 xss + + c ec 1 1 ce C 1+ KM + c + c e E x ss ce c 1 R x ss Michaelis constant (units mol dm -3 ) K C + 1 M 1 Enzyme rate constant (unit : s -1 ) E c C K + c M Catalytic rate constant (unit: s -1 ) Unsaturated enzyme inetics c + c + K E C M C U C τ + E τu s τ C U K C M 1 + Unit: dm 3 mol -1 s -1 Saturated enzyme inetics Time taen for E and S to combine to form productive complex ES Time taen for complex ES to generate and release product 13

14 U K C M ( ) 1 1 K θ 1 K1 θ We consider two sub cases depending on the value of θ. θ >> 1 scenario. 1+ θ θ U K1 1K1 1 RDS 1 E + S -1 ES Unsaturated rate constant reflects Rate determining substrate binding to enzyme. U 1 E + P U K C M ( ) 1 1 K θ 1 K1 θ θ << 1 scenario. 1+θ 1 U K 1 RDS -1 Fast pre-equilibrium followed by a slow rate determining decomposition of the ES complex to form products. 1 E + S ES U K 1 E + P 14

15 When >> -1, ES complex decomposition to form products is faster than ES decomposition bac to reactants. The rds will involve the rate of combination of E and S to form the ES complex. The ES complex will be short lived since it does not accumulate to form products. Unsaturated enzyme inetics. τ U + U 1 1 K1 RDS -1 RDS E + S ES ES E + P E + S E + P Have a fast pre-equilibrium followed by a slow rate determining decomposition of the ES complex to form products. R Enzyme catalysed reaction: Variation of reaction rate with substrate (reactant) concentration. e E c>> K R E c M e c E c C K + c c<< K M M c E c Uc KM R ce U ΨR / c e Unsaturated enzyme Kinetics 1 st order wrt substrate Saturated enzyme Kinetics Zero order wrt substrate uc/k M Rate variation adopts shape of rectangular hyperbola. R u Ψ e 1 + u c 15

16 Single substrate Michaelis-Menten Kinetics. Enzyme-substrate complex Rate Also termed c or turnover number max number of enzymatic reactions catalyzed per second. c /K M measures catalytic efficiency of enzyme. Maximum value corresponds to Diffusion control reaction between S and E in solution (10 10 M -1 s -1 ). 16

17 Determination of inetic parameters K M and c for enzyme catalysis. options: Fit raw rate vs concentration data to MM equation using Non-Linear Least Squares (NLLS) fitting. Transform rate vs concentration data into suitable linear format such as Hanes plot or Lineweaver-Bur plot. Lineweaver-Bur equation ec + C R,0 R,0 ( c KM) e c ce KM + c S c + I LB LB Michaelis-Menten equation 1 S LB K M e C I LB 1 e C R I LB S LB c Plot reciprocal initial reaction rate Versus reciprocal substrate concentration. Measure slope S LB and intercept I LB of plot. 17

18 Redox enzyme systems based on oxygen or peroxide electrochemistry The most commonly used enzymes in the design of enzyme electrodes contain redox groups which change redox state during the biochemical reaction. Enzymes of this type are the oxidases and the pyrroloquinoline quinone (PQQ) dependent dehydrogenases. In nature, oxidase enzymes such as glucose lactate and cholesterol oxidase act by oxidising their substrates, accepting electrons in the process and thereby changing to an inactivated reduced state. These enzymes are normally returned to their active oxidised state by transferring these electrons to molecular oxygen, resulting in the production of hydrogen peroxide (H ). gluconolactone glucose This naturally occurring Ping pong mechanism Gx Gx for the oxidation of FADH FAD + Ping pong mechanism glucose by oxygen can be readily utilized catalysed by glucose in an amperometric oxidase. biosensor device. H Gx Ping-Pong Mechanism S Glucose P Gluconolactone E Gx (oxidized form) FAD + Gx (reduced form) FADH Substrate/enzyme inetics M Enzyme/mediator inetics H Reduced form xidized form Mediated or two substrate enzyme system. + Gx + H β-d-glucose D-glucono-1,5-lactone 18

19 The mechanism of FADH oxidation by the natural electron acceptor. The reaction results in H formation. Amperometric glucose detection Because both oxygen and hydrogen peroxide are both electrochemically active, the progress of the biochemical reaction can be followed by either reducing the oxygen (cosubstrate) or oxidising the hydrogen peroxide (product). The method based upon oxygen reduction at an electrode is one of the simplest but suffers from several disadvantages namely, slow response characteristics, difficulties in miniaturization, low accuracy and reproducibility. Measurements based upon hydrogen peroxide oxidation can overcome these problems and indeed represent by far the most popular approach. A major limitation of the peroxide detection approach is the high operating voltage (circa 0.8 volts vs the Ag/AgCl reference electrode) required to oxidise the hydrogen peroxide resulting in the possibility of interference. The use of mediators (molecules which can shuttle electrons between the redox centre of the enzyme and the electrode) can minimise this problem as they can, depending on the compound used, be regenerated at potentials where interference from species such as ascorbate, urate and paracetamol is minimized. 19

20 Generalized Ping-Pong dual substrate mechanism. S Glucose P Gluconolactone E Substrate/enzyme inetics Michaelis Menten Kinetics M xidized enzyme E X Reduced enzyme E RED Enzyme/mediator inetics M RED Reduced form M X xidized form Have possibility of amperometrically detecting M X or M RED at detector electrode in biosensor device. Redox mediator may be present in solution next to electrode (homogeneous mediation), or be immobilized on electrode surface (heterogeneous mediation). e - M RED M E X E Substrate S Ferrocene Fe(Cp) Ferricinium Fe(Cp) + M X Detector Electrode. Current flow proportional to substrate concentration. G + FAD GL + FADH FADH Fe E RED + + Fe( Cp) FAD + Fe( Cp) + ( Cp) Fe( Cp) + e + H + Product P Mae current measurement In this region. M RED,M X represents reduced and oxidised forms of redox mediator (ferrocene and ferricinium); G glucose, GL gluconolactone. Gx (FADH ) reduced form of glucose oxidase; Gx(FAD) oxidised form of glucose oxidase. 0