Chapter 6: Basic Concepts of Enzyme Action
To understand how enzymes operate, we need to consider only two thermodynamic properties of the reaction:
(1) the free-energy difference (ΔG) between the products and the reactants (this determines whether the reaction will take place spontaneously) (2) the free energy required to initiate the conversion of reactants into products (this determines the rate of the reaction; enzymes only affect (2))
Cofactors can be subdivided into two groups:
1) small organic molecules, derived from vitamins called coenzymes 2) metals
To determine the free-energy change of the reaction, we need to take into account two things:
1) the nature of both the reactants and the products as well as 2) their concentrations.
The free-energy change of a reaction (ΔG) tells us whether the reaction can take place spontaneously: (list the five principles)
1. A reaction can take place spontaneously only if ΔG is negative. 2. A reaction cannot take place spontaneously if ΔG is positive. An input of free energy is required to drive such a reaction. These reactions are termed endergonic. 3. In a system at equilibrium, there is no net change in the concentrations of the products and reactants, and ΔG is zero. 4. The ΔG of a reaction depends only on the free energy of the products (the final state) minus the free energy of the reactants (the initial state). The ΔG of a reaction is independent of the path (or molecular mechanism) of the transformation. The mechanism of a reaction has no effect on ΔG. For example, the ΔG for the transformation of glucose into CO2 and H2O is the same whether it takes place by combustion or by a series of enzyme-catalyzed steps in a cell. 5. The ΔG provides no information about the rate of a reaction. A negative ΔG indicates that a reaction can take place spontaneously, but it does not signify whether it will proceed at a perceptible rate.
The Six Major Classes of Enzymes
1. Oxidoreductases 2. Transferases 3. Hydrolyases 4. Lyases 5. Isomerases 6. Ligases
Although enzymes differ widely in structure, specificity, and mode of catalysis, a number of generalizations concerning their active sites can be stated:
1. The active site is a three-dimensional cleft or crevice formed by groups that come from different parts of the amino acid sequence: indeed, amino acids near to one another in the primary structure are often sterically constrained from adopting the structural relations necessary to form the active site. 2. The active site takes up a small part of the total volume of an enzyme. Although most of the amino acid residues in an enzyme are not in contact with the substrate, the cooperative motions of the enzyme as a whole help to correctly position the catalytic residues at the active site. 3. Active sites are unique microenvironments. The close association between the active site and the substrate means that water is usually excluded from the active site unless it is a reactant. The nonpolar microenvironment of the cleft enhances the binding of substrates as well as catalysis. Nevertheless, the cleft may also contain polar residues, some of which may acquire special properties essential for substrate binding or catalysis. The internal positions of these polar residues are biologically crucial exceptions to the general rule that polar residues are located on the surface of proteins, exposed to water. 4. Substrates are bound to enzymes by multiple weak attractions. The noncovalent interactions between the enzyme and the substrate in ES complexes are much weaker than covalent bonds. These weak reversible interactions are mediated by electrostatic interactions, hydrogen bonds, and van der Waals forces, powered by the hydrophobic effect. 5. The specificity of binding depends on the precisely defined arrangement of atoms in an active site. Because the enzyme and the substrate interact by means of short-range forces that require close contact, a substrate must have a matching shape to fit into the site. Emil Fischer's analogy of the lock and key expressed in 1890, has proved to be highly stimulating and fruitful. However, we now know that enzymes are flexible and that the shapes of the active sites can be markedly modified by the binding of substrate, a process of dynamic recognition called induced fit . Moreover, the substrate may bind to only certain conformations of the enzyme, in what is called conformation selection. Thus, the mechanism of catalysis is dynamic, involving structural changes with multiple intermediates of both reactants and the enzyme.
A kilojoule (kJ) is equal to ______ J. What is a joule (J)? A kilocalorie (kcal) is equal to ______ cal. What is a calorie (cal)? 1 kJ = ______ kcal
1000 the amount of energy needed to apply a 1-newton force over a distance of 1 meter. 1000 It is equivalent to the amount of heat required to raise the temperature of 1 gram of water from 14.5°C to 15.5°C. 0.239
Experimental attempts to reduce the size of a catalytically active enzyme show that the minimum size requires about _____ amino acid residues. In fact, nearly all enzymes are made up of more than _____ amino acid residues, which gives them a mass greater than 10 kDa and a diameter of more than 25 Å, suggesting that
100; 100; all amino acids in the protein, not just those at the active site, are ultimately required to form a functional enzyme.
For carbonic anhydrase: Each enzyme molecule can hydrate _____ molecules of CO2 per second. This catalyzed reaction is _____ times as fast as the uncatalyzed one. The transfer of CO2 from the ______ to the ________ and then to the _________ would be less complete in the absence of this enzyme.
10^6; 10^7; tissues; blood; lungs
A convention has been adopted to simplify free-energy calculations for biochemical reactions. The standard state is defined as having a pH of ___. Consequently, when H+ is a reactant, its concentration has the value ___ (corresponding to a pH of 7) in the numbered equations that follow. The concentration of water also is taken to be __ in these equations
7; 1;1
The peptide catalyzes into what two components?
A carboxyl component and an amino component
Hydrolyases
A hydrolyase cleaves molecules by the addition of water. Trypsin, the proteolytic enzyme already discussed, is a hydrolyase
Lyases
A lyase adds atoms or functional groups to a double bond or removes them to form double bonds. The lyase fumarase is crucial to aerobic fuel metabolism.
Proteolytic Enzymes Illustrate the Range of __________ _________
Enzyme Specificity
The Standard Free-Energy Change of a Reaction Is Related to the ________ _________
Equilibrium Constant
Enzymes lower the activation energy, but where does the energy to lower the activation energy come from?
Free energy is released by the formation of a large number of weak interactions between a complementary enzyme and substrate.
What does the active site of the enzyme contain?
It contains the amino acid residues that directly participate in the making and breaking of bonds. These residues are called the catalytic groups.
Ligases
Ligases join two molecules in a reaction powered by ATP hydrolysis. DNA ligase, an important enzyme in DNA replication, is representative of this class.
Importance of lysozyme
Lysozyme, found in a variety of organisms and tissues including human tears, degrades the cell walls of some bacteria.
Examples?
Most enzymes are named for their substrates and for the reactions that they catalyze, with the suffix "ase" added. Thus, a peptide hydrolase is an enzyme that hydrolyzes peptide bonds, whereas ATP synthase is an enzyme that synthesizes ATP
Use papain, trypsin, and and thrombin to explain how proteolytic enzymes differ markedly in their degree of substrate specificity:
Papain, which is found in papaya plants, is quite undiscriminating: it will cleave any peptide bond with little regard to the identity of the adjacent side chains. This lack of specificity accounts for its use in meat-tenderizing sauces. Trypsin, a digestive enzyme, is quite specific and catalyzes the splitting of peptide bonds only on the carboxyl side of lysine and arginine residues. Thrombin, an enzyme that participates in blood clotting, is even more specific than trypsin. It catalyzes the hydrolysis of Arg-Gly bonds in particular peptide sequences only
The Free-Energy Change Provides Information About the _________ but Not
Spontaneity; the Rate of a Reaction
For the first step:
Substrates bind to a specific region of the enzyme called the active site.
What is the First Step in Enzymatic Catalysis
The Formation of an Enzyme-Substrate Complex
What is the active site of the enzyme?
The active site of an enzyme is the region that binds the substrates (and the cofactor, if any). It is the region of the enzyme that most directly lowers the ΔG‡ of the reaction, thus providing the rate-enhancement characteristic of enzyme action.
Why does the rate of product formation level off with time?
The reaction has reached equilibrium. Substrate S is still being converted into product P, but P is being converted into S at a rate such that the amount of P remains constant.
What is the transition state?
The transition state is a fleeting molecular structure that is no longer the substrate but is not yet the product. The transition state is the least-stable and most-seldom (rare)-occurring species along the reaction pathway because it is the one with the highest free energy.
Isomerases
These enzymes move functional groups within a molecule. We will meet triose phosphate isomerase in glycolysis.
Oxidoreductases
These enzymes transfer electrons between molecules. In other words, these enzymes catalyze oxidation-reduction reactions. We will first meet a member of this class, lactate dehydrogenase, when we consider glycolysis, the first pathway in glucose degradation.
Transferases
These enzymes transfer functional groups between molecules. Aminotransferases are prominent in amino acid synthesis and degradation, where they shuffle amine groups between donor and acceptor molecules.
How can we explain the rate enhancement in terms of thermodynamics?
To do so, we have to consider not the end points of the reaction but the chemical pathway between the end points.
Enzymes Facilitate the Formation of the _______ ________
Transition State
What Are Potent Inhibitors of Enzymes
Transition-State Analogs
Gibbs free energy, or more simply free energy (G), is
a thermodynamic property that is a measure of useful energy, or energy that is capable of doing work
Enzymes ________ the rate of reactions by factors of as much as a ______ or more
accelerate; million
If our understanding of the importance of the transition state to catalysis is correct, then ______ that recognize transition states should function as catalysts.
antibodies
An enzyme without its cofactor is referred to as an __________ ; the complete, catalytically active enzyme is called a _________
apoenzyme; holoenzyme
Indeed, the catalytic specificity of enzymes depends in part on the specificity of ______
binding
The free energy released on binding is called the
binding energy
(Look at equation for this reaction on page 97) Even a reaction as simple as adding water to carbon dioxide is catalyzed by an enzyme—namely _________ ________ . This reaction facilitates
carbonic anhydrase (one of the fastest known enzymes); the transport of carbon dioxide from the tissues where it is produced to the lungs where it is exhaled.
Different enzymes that use the same ______ usually carry out similar chemical transformations.
coenzyme
Many Enzymes Require _______ for Activity
cofactors
ΔG° is the free-energy change for this reaction under standard conditions—that is, when
each of the reactants A, B, C, and D is present at a concentration of 1.0 M (for a gas, the standard state is usually chosen to be 1 atmosphere) before the initiation of the reaction, and the temperature is 298 K (298 kelvins, or 25°C).
The activation energy immediately suggests how
enzymes accelerate the reaction rate without altering ΔG of the reaction
A simple way to determine the ΔG°′ is to measure the concentrations of reactants and products when the reaction has reached ______. At equilibrium, there is ___ net change in the concentrations of reactants and products; in essence,
equilibrium; no; the reaction has stopped and ΔG = 0.
The free-energy difference between reactants and products accounts for the ______ of a reaction, but enzymes accelerate how quickly
equilibrium; this equilibrium is attained
An analog that also carries a negative charge on α-carbon would be expected to bind
even more tightly.
The six groups (classes) of enzymes were subdivided and further subdivided so that a ____-digit number preceded by the letters _____ for _______ _______
four; EC; enzyme commission
Enzymes speed up the rate of chemical reactions, but the properties of the reaction—whether it can take place at all—depends on
free-energy differences
A chemical reaction of substrate S to form product P goes through a transition state X‡ that has a ____ free energy than does either S or P. The double dagger denotes the transition state.
higher
Loosely associated coenzymes are more like cosubstrates because,
like substrates and products, they bind to the enzyme and are released from it.
Enzymes function to _____ the activation energy.
lower; In other words, enzymes facilitate the formation of the transition state.
Although the chemical repertoire of amino acid functional groups is quite varied, they often cannot.... Thus, the catalytic activity of many enzymes depends on the presence of _____ molecules termed ________.The precise role varies with the _______ and the _________.
meet the chemical needs required for catalysis to take place; small; cofactors; cofactor; enzyme
What are transition-state analogs?
mimics that are compounds that resemble the transition state of a reaction but are not capable of being acted on by the enzyme.
The interaction of the enzyme with the substrate and reaction intermediates is fleeting, with
molecular movements resulting in the optimal alignment of functional groups at the active site so that maximum binding energy occurs only between the enzyme and the transition state, the least-stable reaction intermediate.
Because the activation energy is lower,
more molecules have the energy required to reach the transition state and more product will be formed faster
The classification of all enzymes into these six classes also allowed the development of a standard
nomenclature for enzymes
Coenzymes are distinct from normal substrates
not only because they are often derived from vitamins but also because they are used by a variety of enzymes.
Van der Waals forces become significant in binding only when
numerous substrate atoms simultaneously come close to many enzyme atoms. Hence, to bind as strongly as possible, the enzyme and substrate should have complementary shapes.
Enzymes accelerate the attainment of equilibria but do not shift their ______. The equilibrium position is a function only of
positions; the free-energy difference between reactants and products.
Tightly bound coenzymes are called ________ ________
prosthetic (helper) groups.
The biochemical function of proteolytic enzymes is to catalyze
proteolysis, the hydrolysis of a peptide bond
Antibodies have been generated that
recognize the transition states of certain reactions, and these antibodies, called catalytic antibodies or abzymes, do indeed function as enzymes.
The inhibitory power of transition-state analogs underscores the essence of catalysis:
selective binding of the transition state.
The essence of catalysis is
stabilization of the transition state.
Proteolytic enzymes differ markedly in their degree of __________ ________.
substrate specificity
Most enzymes are highly selective in the
substrates that they bind.
In general, highly potent and specific inhibitors of enzymes can be produced by
synthesizing compounds that more closely resemble the transition state than the substrate itself.
"Spontaneously" in the context of thermodynamics means that the reaction will
take place without the input of energy and, in fact, the reaction releases energy. Such reactions are said to be exergonic.
Enzymes Alter the Reaction Rate but Not
the Reaction Equilibrium (An enzyme cannot alter the laws of thermodynamics and consequently cannot alter the equilibrium of a chemical reaction)
It is important to stress that whether the ΔG for a reaction is larger, smaller, or the same as ΔG°′ depends on
the concentrations of the reactants and products
What is racemization?
the conversion of one enantiomer into another; in regard to proline, the interconversion of the L and D isomers.
Note that the energy of activation, or ΔG‡, does not enter into the final ΔG calculation for the reaction, because
the energy that had to be added to reach the transition state is released when the transition state becomes the product.
What is enzyme commission?
the entity that developed the classification system, could precisely identify all enzymes.
Thus, the maximal binding energy is released when
the enzyme facilitates the formation of the transition state.
In essence, the interaction of the enzyme and substrate at the active site promotes
the formation of the transition state.
The rate of a reaction depends on
the free energy of activation (ΔG‡), which is largely unrelated to the ΔG of the reaction.
The difference in free energy between the transition state and the substrate is called
the free energy of activation or simply the activation energy, symbolized by ΔG‡
This precision is a result of
the intricate three-dimensional structure of the enzyme protein.
Thus, the ΔG of a reaction depends on
the nature of the reactants (expressed in the ΔG° term of equation 1) and on their concentrations (expressed in the logarithmic term of equation 1).
The specificity of an enzyme is due to
the precise interaction of the substrate with the enzyme.
Enzymes are highly specific both in
the reactions that they catalyze and in their choice of reactants, which are called substrates.
the full complement of such interactions is formed only when
the substrate is in the transition state
The combination of substrate and enzyme creates a reaction pathway whose _______ ______ _______ is lower than what it would be without the enzyme
transition-state energy
However, the transition state is too _____ to exist for long. It
unstable; collapses to either substrate or product, but which of the two accumulates is determined only by the energy difference between the substrate and the product—that is, by the ΔG of the reaction.
The criterion of spontaneity for a reaction is ___ not ____. This point is important because reactions that are not spontaneous,
ΔG; ΔG°′; on the basis of ΔG°′, can be made spontaneous by adjusting the concentrations of reactants and products. This principle is the basis of the coupling of reactions to form metabolic pathway
For this equation, list what each letter represents:
ΔG° is the standard free-energy change, R is the gas constant, T is the absolute temperature, and [A], [B], [C], and [D] are the molar concentrations of the reactants.
