Lecture 4

1. Lecture 4 Models of enzyme action Dr. NasirJalal ASAB, NUST, H-12 Islamabad

2. Quotes of the day(Samuel Langhorn Clemens) 1835-1910Mark Twain • “I have never let my schooling interfere with my education.” • “Education: the path from cocky ignorance to miserable uncertainty.” ― Mark Twain

3. Models of enzyme action "Lock and key" model : "Lock and key" model Enzymes are very specific, because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve. Induced fit model : Induced fit model Diagrams to show the induced fit hypothesis of enzyme action. since enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site. In some cases, such as glycosidases , the substrate molecule also changes shape slightly as it enters the active site.

4. Transition State Model • Transition State Theory: • In the transition state theory, the mechanism of interaction of reactants is not considered; the important criterion is that colliding molecules must have sufficient energy to overcome a potential energy barrier (the activation energy) to react. • It takes a lot of energy to achieve the transition state, so the state is a high-energy substance. The potential energy of the system increases at this point because: • The approaching reactant molecules must overcome the mutual repulsive forces between the outer shell electrons of their constituent atoms. • Atoms must be separated from each other as bonds are broken.

5. Lock and Key Model + + E + S ES complex E + P P S S P

6. Enzyme Action: Lock and Key Model • An enzyme binds a substrate in a region called the active site • Only certain substrates can fit the active site • Amino acid R groups in the active site help substrate bind • Enzyme-substrate complex forms • Substrate reacts to form product • Product is released

7. Enzyme Action: Induced Fit Model • E + S ES complex E + P P S S S P

8. Enzyme Action: Induced Fit Model • Enzyme structure flexible, not rigid • Enzyme and active site adjust shape to bind substrate • Increases range of substrate specificity • Shape changes also improve catalysis during reaction

9. Why are enzyme kinetics? Dihydrofolatereductase enzyme binds to two substrates (dihydrofolate and NADPH) Studying enzyme kinetics can tell the sequence in which the substrates bind and the sequence in which products are formed. Although these mechanisms are often a complex series of steps, there is typically one rate-determining step that determines the overall kinetics. This rate-determining step may be a chemical reaction or a conformational change of the enzyme or substrates, such as those involved in the release of product(s) from the enzyme.

10. Enzyme Kinetics • Like other catalysts, enzymes do not alter the position of equilibrium between substrates and products. • Unlike uncatalyzed chemical reactions, enzyme-catalyzed reactions display saturation kinetics. • For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate increases linearly with substrate concentration; the enzyme molecules are largely free to catalyze the reaction, and increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter one another.

11. Enzyme Kinetics • At relatively high substrate concentrations, the reaction rate typically approaches the theoretical maximum; the enzyme active sites are almost all occupied and the reaction rate is determined by the turnover rate of enzyme (i.e. how quickly the enzyme can free its active site). • The substrate concentration midway between two limiting cases (minimum and maximum velocity) is denoted by KM. • The two most important kinetic properties of an enzyme are: • how quickly the enzyme becomes saturated with a particular substrate, and • the maximum rate it can achieve. Knowing these properties suggests what an enzyme might do in the cell and can show how the enzyme will respond to changes in these conditions.

12. Michaelis-Menten As larger amounts of substrate are added to a reaction, the available enzyme binding sites become filled to the limit of Vmax. Beyond this limit the enzyme is saturated with substrate and the reaction rate ceases to increase. Vmax Max rate

13. Enzyme kinetics V0 The Michaelis constant KM is experimentally defined as the concentration at which the rate of the enzyme reaction is half Vmax, which can be verified by substituting [S] = Km into the Michaelis–Menten equation and can also be seen graphically.

14. Michaelis–Menten equation The Michaelis–Menten equation describes how the (initial) reaction rate v0 depends on the position of the substrate-binding equilibrium and the rate constant KM. Two crucial assumptions underlie this equation: 1. quasi-steady-state assumption (or pseudo-steady-state hypothesis), namely that the concentration of the substrate-bound enzyme (and hence also the unbound enzyme) changes much more slowly than those of the product and substrate and thus the change over time of the complex can be set to zero. 2. the total enzyme concentration does not change over time. Question: Why is it not possible to measure velocity of enzyme reaction at the initial stage?

15. Michaelis–Menten equation • If [S] is small compared to KM then the term • and very little ES complex is formed, • thus • Therefore, the rate of product formation is • Thus the rate of product formation depends on the enzyme concentration as well as on the substrate concentration.

16. Linear plot of Michaelis-Menten The plot of v versus [S] is not linear; although initially linear at low [S], it bends over to saturate at high [S]. Before the modern era of nonlinear curve-fitting on computers, this nonlinearity could make it difficult to estimate KM and Vmax accurately. Therefore, several researchers developed linearisations of the Michaelis–Menten equation, such as the Lineweaver–Burk plot, the Eadie–Hofstee diagram and the Hanes–Woolf plot. All of these linear representations can be useful for visualising data, but none should be used to determine kinetic parameters, as computer software is readily available that allows for more accurate determination by nonlinear regression methods

17. Lineweaver–Burk plot • The Lineweaver–Burk plot or double reciprocal plot is a common way of illustrating kinetic data. This is produced by taking the reciprocal of both sides of the Michaelis–Menten equation. This is a linear form of the Michaelis–Menten equation and produces a straight line with the equation: • y = mx + c with a y-intercept equivalent to 1/Vmax and an x-intercept of the graph representing -1/KM