I. Cellular Metabolism & Reaction Coupling Figure 1: Metabolism Metabolism represents the sum total of ALL chemical reactions within the cell. These reactions can be regarded as either catabolic or anabolic in nature. The thermodynamic properties of catabolic & anabolic metabolic pathways can be illustrated via an examination of the Gibbs free energy formula: Gibbs Free Energy (G) represents the total amount of energy within a system available to perform work. Changes in the amount of free energy within a system can be calculated with the expression: G = H (T S) H = change in enthalpy (total energy [chemical + heat]) S = change in entropy (disorder -inversely proportional to temperature) G = change free energy available to perform cellular work (i.e. transport, synthesis, etc) Entropy (S) is the most random form of energy, directly proportional to temperature & not useful for performing work. Thus, after eliminating the entropy component of H in a system (T S), the remaining energy ( G) represents what is available to perform cellular work. Changes in the free energy of a system as a result of a metabolic process provides information regarding the spontaneity of a process. A Spontaneous reaction is one in which, one the Ea is satisfied, requires no additional energy input (is a self-sustaining process). It does NOT mean that the process occurs rapidly! 1
Figure 2: Thermodynamic Properties of Metabolic Pathways Catabolic Reactions: involve the breakdown of a molecule into simpler subunits. Examples include hydrolysis, cell respiration, etc. Such reactions tend to be spontaneous, since they involve a net release of energy (- H) & an increase in entropy (+ S). As previously stated, spontaneous reactions are those that, once the initial E a is satisfied, require no additional energy input to occur (are self-sustaining). Anabolic Reactions: involve the coupling of subunits to form a more complex molecule. Examples include dehydration synthesis, DNA replication, etc. Such reactions tend to be nonspontaneous, since they involve a net consumption of energy (+ H) & a decrease in entropy (+ S). In nonliving systems (i.e. test tubes), the likelihood of a reaction s spontaneity can be enhanced by changing the temperature of the system. This is not possible in living systems such as the cell, for the temperature is constant & vigorously regulated. Thus, in order to drive the many nonspontaneous pathways vital to cellular metabolism, they must be coupled (linked) to spontaneous reactions. II. Metabolic Pathways & ATP Figure 3: Non-Spontaneous Metabolic Pathway: Sucrose Synthesis 2
In the above reaction, more energy is consumed in the breaking of bonds than in the making of bonds (+ΔH). In addition, it results in a decrease in the entropy of the surroundings (-ΔS). Therefore, this reaction is Nonspontaneous. The introduction of ATP to this pathway can increase its likelihood of occurring spontaneously Figure 3.1: Spontaneous Metabolic Pathway: ATP Hydrolysis The liberated phosphate (Pi) from ATP hydrolysis can be transferred to another molecule, a process known as Phosphorylation. The resulting Phosphorylated Intermediate is more likely to react exergonically (-ΔH) with other molecules to promote spontaneity. Consider the transfer of (Pi) from ATP to the glucose molecule in the 1st reaction: Figure 3.2: Coupling of Spontaneous & Non-Spontaneous Pathways Glucose + ATP Glucose-6-Phosphate + Fructose Sucrose + H2O + Pi *Phosphorylated Intermediate G = *The tendency of the phosphorylated intermediate to react exergonically with another molecule will contribute to an increase in the system s entropy (+ S). The combined effects of the decrease in enthalpy & increase in entropy increases the likelihood that the pathway will occur spontaneously. Even though ATP makes reactions more likely to be spontaneous, such reaction may not occur at a rate suitable to sustain cell metabolism. Metabolic reactions occur at suitable rates thanks to the action of specialized proteins called Enzymes, which lower the energy requirements (Ea) for reactions to occur. 3
III. Enzymes & Activation Energy (Ea) Figure 4: Free Energy of Activation Activation Energy: a) The E a required for a given reaction is usually provided in the form of heat absorbed by the reactants from the surroundings. As heat is absorbed, the reactant molecules collide more frequently & forcefully (this thermal agitation makes the bonds more likely to break, allowing new ones to form). b) For some reactions, the E a is low enough that there is sufficient thermal energy at room temperature for many of the reactants to reach the transition state & react. As a result, these reactions occur very quickly. For others, E a is so high it occurs very slowly, for only a small percentage of the molecules overcome the E a & will only occur if the reactants are heated. Effects of Enzymes on Activation Energy The amount of heat energy required to overcome the E a barrier for many reactions would likely kill the cell. Thus organisms employ specialized proteins called Enzymes to overcome this barrier so vital reactions may occur at temperatures that would not threaten the cell. Figure 5: Generalized Enzyme Structure 4
Enzymes are organic catalysts whose Active Sites are substrate-specific & exhibit the following features: a) The shape of the active site is complementary to its native substrate b) The active site promotes the formation of and / or breaking of bonds within reactant molecules. c) Some enzymes possess multiple active sites for a given substrate. d) Allosteric Sites can bind to molecules that can regulate (inhibit/active) enzyme activity. e) The enzyme is NEVER permanently altered as a result of interacting with its substrate. Figure 5.1: Effects of Enzymes on Ea Requirements Combined Effects: Figure 5.2: Enzyme Catalyzed Reactions 5
Enzyme Binding Models There exists two models / hypotheses to explain the interaction between an enzyme s active site & a substrate molecule: the lock and key & induced fit model Figure 6: Enzyme Binding Models: Lock & Key Lock & Key Model Flaws Lock & Key Model: the enzyme s active site is rigid with a fixed shape. Therefore, the active site, much like a key, exactly matches the shape of a particular substrate, the lock. Not only must the enzyme & substrate be complementary in terms of geometry, but electrically as well to avoid repulsive forces. It is now known, through experimentation, that the active sites of many enzymes do NOT have a shape that exactly matches its substrate. To explain this observation, a new enzyme binding model was proposed Figure 6.1: Enzyme Binding Models: Induced Fit Induced Fit Model: the enzyme s active site is flexible, whereupon it bends slightly upon accepting its substrate. This brings catalytic & functional groups lining the active site into the proper orientation to react with the substrate. 6
Figure 6.2: Enzyme Specificity w/in Induced Fit Model Within the context of the induced fit model, the specificity of the enzyme to a substrate stems from the fact that some substrates can cause the active site to bend too much or too little to adversely affect the enzyme s function. Comparison of Enzyme Structures Figure 7: Simple vs Complex Enzymes Simple Enzyme Complex Enzyme Simple Enzymes: are composed of a single protein (e.g. pepsin, a protein degrading enzyme formed in the stomach). Complex Enzymes: are composed of a protein portion, the Apoenzyme, & a non-protein Prosthetic Group that may be either a Coenzyme or Cofactor. The apoenzyme is NOT CATALYTIC without its specific prosthetic group... Figure 7.1: Complex Enzyme Prosthetic Groups: Coenzymes Coenzymes: 7
Figure 7.2: Complex Enzyme Prosthetic Groups: Cofactors Cofactors are inorganic prosthetic groups having a metal ion bound to a porphyrin ring chelator. Heme Groups are enzyme / protein cofactors adapted for binding O 2 (heme of Hb) or photons (heme of chlorophyll). Figure 7.3: Heme Group Cofactor of Hb Enzyme Cofactors: IV. Enzyme Kinetics The study of the amount of product that can be produced by an enzyme per unit time is called Enzyme Kinetics. There are several environmental factors (ph, temperature, substrate concentration) that influence enzyme kinetics. Consider an increasing concentration of substrate added to tubes containing a FIXED concentration of enzyme. Assume that the conditions of temperature & ph are the SAME for each tube. The initial velocity of reaction [V0] is measured for each tube (product formed / time) & plotted 8
Figure 8: Michaelis-Menton Enzyme Kinetics When plotted, the V 0 s of each tube generate a RECTANGULAR HYPEBOLA called the Michaelis-Menton curve. Figure 8.1: Changes in V 0 w/increasing Substrate Concentration Since enzyme concentration is a limiting factor in the reaction, increasing [S] doesn t result in a proportional increase in V 0. In other words, at high [S], there are more substrates than the enzymes can react with. At high [S] concentrations, [V] is unaffected by increasing [S], for a single enzyme can only react w/one substrate at a time. Likewise, the number of cars that can pass through a tollbooth is unaffected by the number of cars waiting, but rather the number of tollbooths that are open. 9
Figure 8.2: Vmax & Enzyme Concentration At high [S], Vmax for a reaction is dependent on the concentration of enzyme & thus is NOT A CONSTANT. Therefore, Vmax is not a good indicator of how efficient an enzyme is at catalyzing a reaction. Figure 8.3: Enzyme-Substrate Affinity: Km As indicated in figure 8.3, the Km value is a constant representing the substrate concentration needed to reach ½ Vmax. The lower the substrate concentration required to reach ½ Vmax, the greater the enzyme s active site is attracted to its specific substrate(s). Thus, low Km values imply a high affinity between enzymes & their substrates & vice-versa. 10
Figure 8.4: Comparing Km Values As indicated in figure 8.4, the km of enzyme 1 is approximately.18 compared to that of enzyme 2, which is approximately.44. What do these km values suggest regarding each enzymes affinity for its particular substrate? Figure 9: Sigmoid Enzyme Kinetics: Allosteric Enzymes Many enzymes are Allosteric, consisting of multiple subunits, each w/an active site for the same substrate. The binding of a single substrate causes a conformational (shape) change in the other subunits, activating them. This initiates a *positive feedback mechanism by which many substrates can be rapidly bound. *positive feedback involes an event that is reenforced by its own outcome (binding of substrate results in binding of even MORE substrate). 11
Figure 9.1: Sigmoid Enzyme Kinetics Curve Allosteric enzymes produce a Sigmoidal (s-shaped) curve when V 0 is plotted against [S]. V 0 is low at low [S], then rapidly increases at higher [S] until Vmax is reached (saturation). V. Nonspecific Factors Affecting Enzyme Kinetics Figure 10: Temperature Effects As seen in figure 10, as temperature rises toward the optimum, the rate of enzyme-induced reaction gradually increases. Once an optimum temperature is reached, the reaction rate reaches Vmax. If temperatures increase past the optimum, the reaction rate will crash due to disruption of the bonds contributing to the enzyme s shape, a condition called denaturation. Human enzymes work best at body temperature (37C). 12
Figure 10.1: Temperature Effects on Hemoglobin As hemoglobin (Hb) approaches body tissue, the temperature of the blood rises (metabolically active tissue produces heat as a by-product). This slight rise in temperature causes conformational changes in the Hb subunits such that they lose affinity for O 2. As a result, O 2 is released, by which it diffuses from the blood into the cells of body tissue. Figure 11: ph Effects All enzymes work best at an optimum ph. Most human enzymes work best at or near a ph of 7, although digestive enzymes in the stomach (e.g. pepsin) work best at lower ph s that would otherwise denature other enzymes. 13
Figure 11.1: ph Effects on Hemoglobin: Bohr Shift As hemoglobin (Hb) approaches body tissue, the ph of the blood drops (metabolically active tissue produces CO 2 carbonic acid as a by-product). This slight drop in ph causes conformational changes in the Hb subunits such that they lose affinity for O 2. As a result, O 2 is released, by which it diffuses from the blood into the cells of body tissue. Mechanisms of Enzyme Inhibition Figure 12: Competitive Enzyme Inhibition Competitive Inhibitors are similar to the native substrate & binds to & blocks the enzyme s active site. The reduced reaction rate can be reversed by increasing the concentration of the native substrate. 14
Figure 13: Non-Competitive / Allosteric Enzyme Inhibition Allosteric Inhibitors bind tightly to an allosteric site on the enzyme & alters the active site s shape. This reduces the enzyme s affinity to bind to the native substrate, reducing reaction rates. Figure 13.1: Noncompetitive Inhibition & Negative Feedback Control General Mechanism Specific Example 15
Mechanisms of Enzyme Activation Figure 14: Allosteric Enzyme Activators Figure 14.1: Effects of Allosteric Inhibitors & Activators on Reaction Rates *As can be seen in figure 14.1, the km value (amount of substrate required to reach ½ Vmax) is much less when an activators is present than when an inhibitor is present. 16
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