(a small digression before talking about enzymes)Given a reaction: aA + bB ´ cC + dD
How do we decide whether it will proceed spontaneously in the forward direction, the reverse direction or neither (at equilibrium)?
A. 2nd Law of Thermodynamics -- says that for any spontaneous process, the entropy (disorder) of the universe, DSuniverse, must increase. This is very difficult to measure, however ===> we need a criterion of spontaneity which applies to our system (organism).
B. At constant Temperature and Pressure (conditions under which we exist, more or less) the change in Gibbs Free Energy, D G, is this criterion; it is determined by the entropy change of the system, D S, and by the change in enthalpy, D H, or heat content of the system (this is the energy exchanged with the surroundings; if DH < 0 --> the reaction is exothermic and heat is given; if D H > 0 --> the reaction is endothermic and heat is absorbed):
1. DG <0 -- The net reaction will be in the foreward direction
2. D G >0 -- The net reaction will be in the reverse direction
3. D G =0 -- The reaction is at equilibrium, and no net change in either direction occurs
Note: We talk about the Net Reaction because all reactions proceed simultaneously in both directions; even at equilibrium, a chemical reaction is a dynamic process with the rate of the foreward reaction equal to the rate of the reverse reaction so that no "net" reaction takes place.
C. What does DG depend upon? For a given reaction, aA + bB === cC + dD, it depends upon:
1. The nature of the products and reactants ===> DG°, the change in Free Energy when all are in the Standard State (1 M Concentration) ===> Standard Free Energy Change
2. The concentrations of products and reactants, R T ln [C]c[D]d / [A]a[B]b
3. Thus,
R = Ideal Gas Const. (1.99 cal mole-1 deg-1); T is the Absolute Temperature (°Kelvin)
4. A reaction for which DG° > 0 can proceed spontaneously to the right if the concentration term is sufficiently negative (i.e. [C]c[D]d is small and/or [A]a[B]b is large).
5. The reaction will proceed in the indicated direction until D G = 0, at this point the reaction is at Equilibrium; the forward reaction rate equals the back reaction rate:
At equilibrium, DG = 0 and ===>DG° = - R T ln [C]c[D]d / [A]a[B]b = - R T ln Keq
Thus, DG° is just another way of expressing Keq, the tendency of a reaction to proceed as written.
6. NOTE--A reaction may proceed spontaneously if:
a) DG° > 0 -- DG is the relevant parameter. Concentrations have a strong effect
it's the sum DG°' + RT ln [C]c [D]d / [A]a [B]b which is important.b) DS < 0 -- may be counterbalanced by DH
c) DH > 0 -- (heat is absorbed during the reaction) may be counterbalanced by DS.
7. Biochemical reactions frequently involve H+ and take place at [H+] = 10-7M (pH=7) ===> Biochemists use a modified DG° corrected for [H+] = 10-7M rather than 1M, this is called DG°'
8. The catabolic reactions of metabolism (reactions which break down food molecules) extract Free Energy from these food molecules and store it in chemical form. This chemical energy is stored in chemical bonds of certain molecules; the most important is Adenosine Tri- Phosphate, ATP. This type of compound is called a Nucleotide, it consists of a cyclic nitrogen-containing base bonded via an N-glycosidic Bond to a sugar, ribose, (this unit is called a Nucleoside) plus one or more phosphate groups ==> Nucleotide.
Figure 6.8
The phosphoric acid anhydride bonds are often called high energy bonds because they are weak bonds, they have high potential energy. When they are broken followed by formation of stronger bonds (e.g. when H2O hydrolyzes the bond) the products have less free energy than the reactants (DG < 0) and the difference in free energy can be used by cells to do work.
At actual intracellular concentrations, DG = -10 to -14 kcal/mole. This is energy which the cell can use to do work via enzymes. The second phosphoric acid anhydride bond may also be hydrolyzed when more energy is required, but this is less common. The phosphoric acid ester bond contains less energy and is not used in energy-requiring reactions.
9. Energy coupling by phosphate transfer: Figure 6.10
ATP is used by enzymes as a source of free energy to make less favorable reactions occur, this process is called energy coupling because the free energy from ATP hydrolysis is coupled to the energy requiring process of another chemical reaction. An example of this process is the enzyme, glutamine synthetase, which synthesizes the amino acid glutamine by forming an amide from the carboxylic acid group of glutamate's side chain and a molecule of ammonia.
The enzyme, glutamine synthetase, couples this reaction with the hydrolysis of ATP by first creating a phosphorylated intermediate of glutamate with the phosphate group from ATP bound to the carboxyl group of the glutamate sidechain. The enzyme then catalyzes the displacement of the phosphate group by the ammonia molecule. Note that the sum of the two reactions has a favorable negative change in free energy.
1. True catalysts-accelerate chemical reactions but are not consumed in the reactions
2. Proteins--most enzymes are proteins but some RNA molecules catalyze biochemical reactions
3. Often use small molecules as cofactors.
a) Inorganic Ions--Fe++ or Fe+++, Cu++, Mg++, Mn++, Zn++ etc.
b) Organic Molecules--These are often called coenzymes; they carry specific atoms or functional groups which can perform a special function which amino acids cannot. When a cofactor is very tighly bound, it may be called a prosthetic group
B. Nomenclature/Classification of enzymes is based upon the reaction they catalyze. e.g. urease--hydrolysis of urea or arginase--hydrolysis of arginine. suffix "-ase" ===> enzyme:
A few enzymes, however, don't have an -ase suffix; e.g. trypsin, pepsin, chymotrypsin
1. Increase rates (alter kinetics). They don't change thermodynamics (position of equilibrium) ===> enzymes increase the rate of approach to equilibrium. Overall change in free energy (related to Keq) is the same for both ===> position of equilibrium is unchanged Enzymes reduce the energy barrier between reactants and products (may find another reaction pathway with several smaller energy barriers), but don't change the difference in free energy between them:
2. Specificity--determined by substrate binding site. May be very specific (i.e. will only bind one molecule) or may be relatively non-specific (will bind a variety of related molecules).
3. Induced Fit -- Enzyme adapts to bind Substrate and bend it into the transition state whose structure is intermediate between the structures of the substrate and the product.
4. pH Optimum -- since enzymes are composed of aa's, their structures and therefore activity (rate at which they catalyze reactions) depend upon the pH which can drastically alter their structures.
5. Temperature -- rates of enzyme catalyzed reactions increase with temperature (as do all reactions) up the the point where the enzyme's tertiary structure is destroyed (denatured) by heat energy.
Measure initial velocity, vo (rate when very little P has formed) at various [S]
vo -- usually has units of µmoles/min and is measured at 25° C
1.Saturation, at high [S] rate reaches a maximum,
Vmax,
and further increases of [S] don't increase
vo
2. Km (the Michaelis constant) has units of M (molarity) and its value will be similar to the [S] found in the cell
an important subject in metabolism, how are enzymes controlled?
1. Enzyme Inhibition -- Inhibitors can bind the the enzyme and slow it down or stop it completely.
2. Allosteric Proteins -- multisubunit proteins which have active and inactive forms. The two forms are in equilibrium with one another and this equilibrium can be shifted by other molecules
a) substrates and activator molecules bind to the enzyme and stabilize the active form of the enzyme
b) inhibitors stabilize the inactive form.
