Enzymes 2 (2023).pdf

WEEK 13 MPharm Programme Enzymes 2 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Enzymes WEEK Factors That Affect Enzyme Function 13 Effect of pH Changes in pH add or remove H+  small changes in the charges on the enzyme & substrate mole...

WEEK 13 MPharm Programme Enzymes 2 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA112 Enzymes WEEK Factors That Affect Enzyme Function 13 Effect of pH Changes in pH add or remove H+  small changes in the charges on the enzyme & substrate molecules (altered critical ionization states) affect the binding of the substrate with the enzyme active site Optimum pH – peak effect on enzyme-catalysed reaction pH 6-8 for most human enzymes – depends on localised conditions e.g. pepsin (stomach) = pH 2-3; trypsin (small intestines) = pH 8 Extreme pH levels  enzyme denaturation disrupt attraction between charged amino acids disrupt bonds & the enzyme’s 3D shape active site is distorted  loss of substrate fit Slide 37 of 69 MPharm PHA112 Enzymes WEEK Effect of pH on Enzyme Function 13 Optimum pH trypsin Reaction rate pepsin pepsin trypsin 0 1 2 3 4 5 6 7 8 9 10 11 12 pH Slide 38 of 69 MPharm PHA112 Enzymes 13 14 WEEK Factors That Affect Enzyme Function 13 Effect of Salinity (Salt Concentration) Changes in salinity add or remove cations & anions Extreme salinity  enzyme denaturation Enzymes are intolerant of extreme salinity disrupts attraction between charged amino acids affects 2° & 3° enzyme structure Reaction rate disrupts bonds & the enzyme’s 3D shape Salt concentration Slide 39 of 69 MPharm PHA112 Enzymes WEEK Enzyme Kinetics 13 Enzyme kinetics is the study of the rates of chemical reactions that are catalysed by enzymes It provides insight into the mechanisms of enzyme catalysis & their role in metabolism how the activity of enzymes is controlled in the cell how drugs and poisons can inhibit or modulate the activity of enzymes In 1913, Michaelis and Menten proposed the model known as Michaelis-Menten Kinetics to account for & explain how enzymes can increase the rate of metabolic reactions how the reaction rates depend on the concentration of enzyme & substrate Slide 40 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 All enzymes show a ‘saturation effect’ with their substrates At low substrate concentration [S], the reaction rate or velocity, V, is proportional to [S] However, as [S] is increased, the reaction rate falls off, and is no longer proportional to [S] On further increase in [S], the reaction rate or velocity becomes constant and independent of [S] At this stage, the enzyme is saturated with substrate A plot of initial reaction velocity, V, against substrate concentration, [S], gives a rectangular hyperbola The Michaelis-Menten equation or kinetics model was developed to explain or account for this ‘saturation effect’ Slide 41 of 69 MPharm PHA112 Enzymes WEEK 13 Relationship between Reaction Velocity & Substrate Concentration Slide 42 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 Michaelis and Menten proposed the following mechanism for a saturating enzyme-catalysed single substrate reaction: According to this postulate/scheme In an enzyme-catalysed reaction, a free enzyme, E, binds its substrate, S, to form an enzyme-substrate complex, ES ES either undergoes further transformation to yield a final product, P, and the free enzyme, E, or breakdowns via a reverse reaction to form the free enzyme, E and substrate, S Slide 43 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 Rate of formation of ES = k1[E][S] Rate of breakdown of [ES] = k-1[ES] + k2[ES] = (k-1 + k2)[ES] At equilibrium, k1[E][S] = (k-1 + k2)[ES] Re-arranging, [ES] = [E][S]/{(k-1 + k2)/k1} But (k-1 + k2)/k1 = KM (Michaelis constant) Therefore, [ES] = [E][S]/KM Slide 44 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Equation 13 By re-arranging the equations, and making several assumptions, they derived the Michaelis-Menten Equation Vmax S v= K M + S Where, Vmax = the maximum velocity or rate of reaction, at maximum (saturating) concentrations of the substrate KM = (k-1 + k2)/k1 = substrate concentration at which the reaction velocity is 50% of the Vmax (Michaelis constant) [S] = concentration of the substrate, S Slide 45 of 69 MPharm PHA112 Enzymes WEEK Michaelis-Menten Kinetics 13 A graph of initial reaction velocity, V0, against substrate concentration, [S], results in a rectangular curve, where Vmax represents the maximum reaction velocity Slide 46 of 69 MPharm PHA112 Enzymes Linear Transformations of Michaelis-Menten Equation WEEK 13 It is not easy to accurately determine Vmax at high substrate concentrations from the Michaelis-Menten curve Algebraic transformation of the Michaelis-Menten equation into linear forms for plotting experimental data Lineweaver-Burk Plot (also called Double Reciprocal plot) Eadie-Hofstee Plot Slide 47 of 69 MPharm PHA112 Enzymes WEEK Lineweaver-Burk Plot 13 Also called the Double Reciprocal Plot – derived by taking the reciprocal of both sides of the Michaelis-Menten equation, and separating out the components of the numerator on the right side of the equation: 1 1  KM = + v vmax  vmax Slide 48 of 69  1   [ S ]0 MPharm PHA112 Enzymes WEEK Eadie-Hofstee Plot 13 Derived by inverting the Michaelis-Menten equation, and multiplying both sides of the equation by Vmax A plot of V against V/[S] yields Vmax as the y-intercept, Vmax/Km as the x-intercept, and Km as the negative slope v v = −KM + vmax [S ]0 Slide 49 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of KM (Michaelis Constant) It has same unit as the substrate (M) The substrate concentration [S] at which the reaction proceeds at half maximal velocity (50%), i.e. KM = [S] at ½ Vmax A measure of an enzyme’s affinity for its substrate – the lower the KM value, the higher the enzyme’s affinity for the substrate and vice versa Provides an idea of the strength of binding of the substrate to the enzyme molecule – the lower the KM value, the more tightly bound the substrate is to the enzyme for the reaction to be catalysed Indicates the lowest concentration of the substrate [S] the enzyme can recognise before reaction catalysis can occur Describes the substrate concentration at which half the enzyme's active sites are occupied by substrate Slide 50 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of KM (Michaelis Constant) KM Values for Some Key Enzymes Slide 51 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of Vmax Gives an idea of how fast the reaction can occur under ideal circumstances Reveals the turnover number of an enzyme, i.e. the number of substrate molecules being catalysed per second Magnitude varies considerably – from  10 in the case of lysozyme to 600,000 in the case of carbonic anhydrase Slide 52 of 69 MPharm PHA112 Enzymes WEEK 13 Significance of Vmax Turnover Numbers of Some Key Enzymes Slide 53 of 69 MPharm PHA112 Enzymes WEEK Enzyme Inhibition 13 Enzymes are required for most of the processes required for life They catalyse biological reactions by reducing the activation energy needed for the reactions to occur They bind to specific substrates in the reaction pathway & speed up the reaction, but are released unchanged to be used again Activity of enzymes needs to be tightly regulated to maintain homeostasis Regulation is accomplished via enzyme inhibition Enzyme inhibition is widely exploited in clinical therapeutics Slide 54 of 69 MPharm PHA112 Enzymes WEEK Types of Enzyme Inhibition 13 Two types of inhibition Irreversible Reversible Reversible inhibition Competitive Non-competitive Uncompetitive Mixed Slide 55 of 69 MPharm PHA112 Enzymes WEEK Competitive Inhibition 13 The inhibitor (I) is structurally similar to the substrate (S) The inhibitor (I) competes with the substrate (S) for the substrate binding site The inhibitor has virtually no affinity for the enzymesubstrate complex (ES), since the substrate (S) already occupies the inhibitor binding site when bound In the presence of an effective concentration of inhibitor the apparent KM is increased there is no change in the Vmax The inhibition can be reversed by increasing the concentration of the substrate (S) Slide 56 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 57 of 69 Competitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 58 of 69 Competitive Inhibition MPharm PHA112 Enzymes Non-competitive Inhibition WEEK 13 The inhibitor has similar affinity for both the free enzyme (E) and the enzyme-substrate complex (ES) The inhibitor may bind with the free enzyme as well as the enzyme-substrate complex The inhibitor binds with the enzyme at a site which is distinct from the substrate binding site The binding of the inhibitor does not affect the substrate binding, and vice versa However, the inhibitor binding to enzyme or enzymesubstrate complex prevents enzyme from forming its product there is no effect on, or change, in the KM the Vmax for the reaction is decreased Slide 59 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 60 of 69 Non-competitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 61 of 69 Non-competitive Inhibition MPharm PHA112 Enzymes WEEK Uncompetitive Inhibition 13 The inhibitor has affinity for the enzyme-substrate complex (ES), but not the free enzyme (E) The inhibitor binds only the enzyme-substrate complex (ES), but not the free enzyme (E) An inactive ESI complex is formed when the inhibitor reversibly binds to the enzyme-substrate complex The inactive ESI complex does not form a product (P) the apparent KM is decreased – due to the selective binding of the inhibitor to the enzyme-substrate complex (ES) the Vmax for the reaction is also decreased inhibition cannot be reversed by increasing the substrate concentration Slide 62 of 69 MPharm PHA112 Enzymes WEEK 13 Slide 63 of 69 Uncompetitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 64 of 69 Uncompetitive Inhibition MPharm PHA112 Enzymes WEEK 13 Slide 65 of 69 MPharm PHA112 Enzymes WEEK 13 Summary of the Effect of Enzyme Inhibition on the KM and Vmax Type of Inhibition Apparent KM Apparent Vmax Increased Unchanged Competitive Non-competitive Unchanged Uncompetitive Decreased Slide 66 of 69 MPharm PHA112 Decreased Decreased Enzymes WEEK 13 The Inhibitor Constant (Ki) A measure of the affinity of an inhibitor drug for an enzyme Ki values are used to characterize and compare the effectiveness of inhibitors relative to KM Useful and important in evaluating the potential therapeutic value of inhibitor drugs for a given enzyme reaction In general, the lower the Ki value, the tighter the binding, and hence the more effective an inhibitor is Slide 67 of 69 MPharm PHA112 Enzymes WEEK Graphical Determination of the Inhibitor Constant (Ki) 13 Non-competitive inhibitor Slide 68 of 69 MPharm PHA112 Competitive inhibitor Enzymes WEEK 13 Enzymes as Important Drug Targets Disease Enzyme Drug Mechanism Hypertension Angiotensin Converting Enzyme (ACE) Lisinopril Inhibitor Alzheimer’s Disease Acetylcholinesterase (AChE) Donepezil Inhibitor Parkinson’s Disease Inflammation Dopamine β-hydroxylase L-DOPA Substrate Cyclooxygenase 2 (COX 2) NSAIDs, e.g. aspirin, ibuprofen, naproxen, celecoxib Inhibitors Gout Parkinson’s Disease Xanthine oxidase Allopurinol Inhibitor Monoamine oxidase Selegiline Moclobemide Inhibitor Statins Inhibitor Cardiovascular HMG CoA reductase disease Slide 69 of 69 MPharm PHA112 Enzymes