MCBG Session 6 - Enzymes Flashcards
Outline the role of enzymes.
- Enzymes are protein catalysts that increase the velocity of a chemical reaction, and are not consumed during the reaction.
- Virtually all reactions in the body are mediated by enzymes
Outline the properties of enzymes in light of the active site.
- Enzyme molecules contain a special pocket or cleft called the active site.
- The active site contains amino acid side chains that participate in substrate binding and catalysis.
- The substrate binds the enzyme, forming an enzyme–substrate (ES) complex.
- Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis.
- ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product.
Outline the properties of enzymes in light of its specificity.
- Specificity: Enzymes are highly specific, interacting with one or a few substrates and catalysing only one type of chemical reaction.
- The set of enzymes made in a cell determines which metabolic pathways occur in that cell.
Outline the properties of enzymes in light of it regulation.
Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need.
Outline the properties of enzymes in light of its location within the cell.
- Many enzymes are localized in specific organelles within the cell.
- Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions.
- This provides a favourable environment for the reaction, and organizes the thousands of enzymes present in the cell into purposeful pathways.
Outline the properties of enzymes in terms of holoenzymes.
- Some enzymes require molecules other than proteins for enzymic activity.
- The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive.
- If the nonprotein moiety is a metal ion such as Zn2+ or Fe2+, it is called a cofactor.
- If it is a small organic molecule, it is termed a coenzyme. - Coenzymes that only transiently associate with the enzyme are called cosubstrates.
- If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (e.g. FAD).
- Coenzymes frequently are derived from vitamins. E.g. NAD+ contains niacin and FAD contains riboflavin.
Outline how enzymes work in terms of free energy of activation.
- Free energy of activation: The peak of energy in Figure 5.4 is the difference in free energy between the reactant and transition state, where the high-energy intermediate is formed during the conversion of reactant to product.
- Because of the high free energy of activation, the rates of uncatalyzed chemical reactions are often slow.
Outline how enzymes work in terms of rate of reaction.
- Rate of reaction: For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state.
- In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product.
- The rate of reaction is determined by the number of such energized molecules.
- In general, the lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate of the reaction.
Outine how enzymes work in terms of an alternate reaction pathway.
- Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation.
- The enzyme does not change the free energies of the reactants or products and, therefore, does not change the equilibrium of the reaction. It does, however, accelerate the rate with which equilibrium is reached.
Discuss the effect of substrate concentration of reaction velocity in light of the following:
- Maximal velocity
- Hyperbolic shape of enzyme kinetics curve
- Maximal velocity: The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time; velocity is usually expressed as μmol of product formed per minute.
- The rate of an enzyme-catalysed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached.
- The levelling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present.
- Hyperbolic shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten kinetics, in which the plot of initial reaction velocity (V0) against substrate concentration ([S]), is hyperbolic.
- In contrast, allosteric enzymes do not follow Michaelis-Menton kinetics and show a sigmoidal curve that is similar in shape to the oxygen dissociation curve of haemoglobin.
Discuss the effect of temperature on reaction velocity.
- Increase of velocity with temperature: The reaction velocity increases with temperature until a peak velocity is reached.
- This increase is the result of the increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction.
- Decrease of velocity with higher temperature: Further elevation of the temperature results in a decrease in reaction velocity because of temperature-induced denaturation of the enzyme.
- The optimum temperature for most human enzymes is between 35 and 40°C.
- Human enzymes start to denature at temperatures above 40°C, but thermophilic bacteria found in the hot springs have optimum temperatures of 70°C.
Discuss the effect of pH on reaction velocity in light of:
- The effect of pH on the ionisation state of the active site
- The effect of pH on enzyme denaturation.
- Effect of pH on the ionisation of the active site: The concentration of H+ affects reaction velocity in several ways.
- First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or un-ionised state to interact.
- For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (–NH3+).
- At alkaline pH, this group is deprotonated, and the rate of the reaction, therefore, declines.
- Effect of pH on enzyme denaturation: Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains.
- The pH optimum varies for different enzymes: The pH at which maximal enzyme activity is achieved is different for different enzymes, and often reflects the [H+] at which the enzyme functions in the body.
Outline the reaction model.
- where S is the substrate
- E is the enzyme
- ES is the enzyme–substrate complex
- P is the product
- k1, k-1, and k2 are rate constants
Outline the Michaelis-Menten equation.
- where vo = initial reaction velocity
- Vmax = maximal velocity
- Km = Michaelis constant = (k-1 + k2)/k1
- [S] = substrate concentration
An important conclusion of Michaelis-Menten kinetics is the characteristics of Km. Outline this.
- Km—the Michaelis constant—is characteristic of an enzyme and its particular substrate, and reflects the affinity of the enzyme for that substrate.
- Km is numerically equal to the substrate concentration at which the reaction velocity is equal to 1⁄2Vmax. Km does not vary with the concentration of enzyme.
- Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is 1⁄2Vmax .
- Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme.
An important conclusion of Michaelis-Menten kinetics is the relationship of velocity to enzyme concentration. Outline this.
- Relationship of velocity to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations.
- For example, if the enzyme concentration is halved, the initial rate of the reaction (vo), as well as that of Vmax, are reduced to half that of the original.
Outline the Lineweaver-Burk plot.
- When vo is plotted against [S], it is not always possible to determine when Vmax has been achieved, because of the gradual upward slope of the hyperbolic curve at high substrate concentrations.
- However, if 1/vo is plotted versus 1/[S], a straight line is obtained.
- This plot, the Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and Vmax, as well as to determine the mechanism of action of enzyme inhibitors.
- The equation describing the Lineweaver-Burk plot is attached - where the intercept on the x-axis is equal to −1/Km, and the intercept on the y-axis is equal to 1/Vmax
Outline competitive inhibition.
- This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site.
- Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor.
- Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve 1⁄2Vmax.
- Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because -1/Km moves closer to zero from a negative value
Outline non-competitive inhibition.
- This type of inhibition is recognized by its characteristic effect on Vmax. Non-competitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The non-competitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring.
- Effect on Vmax: Non-competitive inhibition cannot be overcome by increasing the concentration of substrate. Thus, non-competitive inhibitors decrease the apparent Vmax of the reaction.
- Effect on Km: Non-competitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the non-competitive inhibitor.
- Effect on Lineweaver-Burk plot: Non-competitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a non-competitive inhibitor, whereas Km is unchanged.
What are amyloid fibres?
Amyloid fibres: the misfolded, insoluble form of a normally soluble protein. They are highly ordered with a high degree of b-sheet. The core b-sheet forms before the rest of the protein. Inter-chain assembly stabilised by hydrophobic interactions between aromatic amino acids.
