Biochemistry-CH6
INCOMPLETE/UNSORTEDHome Intersection Biochemistry
191 of 233 6.1 Pasteur (ferments&fermentation)-->Buchner (yeasts&fermentation)-->Kuhne (enzymes&fermentation)-->Sumner(crystallizaiton&enzymes) MOST ENZYMES ARE PROTEINS Activity depends on stability of native conf - denatured = NO FUNC 1pri, 2sec, 3ter, 4qua are essential to catalytic activity MW (12000-1X106) Enzymes may require and additional chemical component (can require BOTH): Cofactor: requiring an additional one or more inorganic ions Coenzyme: requiring an additional complex organic or metallorganic molecule FUNC - transient carriers of specific fGRPS Prosthetic Group: coenzyme or metal ion that is very tightly/COV bound to enzyme Holoenzyme: Complete, catalytically active enzyme w/ coenzyme or metal ions Apoenzyme/Apoprotein: protein from such holoenzyme
Enzyme Classficaiton - 6 Classes Oxidoreductase Transferase Hydrolase Lyases Isomerase Ligase
Each is assigned a four part classification number and systematic name EX ATP - glucose phosphotransferase - catalyzes the transfer of phosphoryl group from ATP to glucose EC # = 2.7.1.1 First # - (2) Class name - transferase Second # - (7) Subclass - phosphotransferase Third # - (1) Phosphotransferase with OH group - acceptor Fourth # - (1) D-glucose = phosphoryl group acceptor Trivial Name - Hexokinase
6.2 S - substrate P - products Uncatalyzed RXNs tend to be slow Most biomols are stable in 7pH, mild T and aq environment Many common RXNs = unfavorable/unlikely in cellular env Active site (aSITE): (dist feature of enzyme) RXN takes place w/in pocket on enzyme Surface is lined with AA residues w/ substituent groups that bind the substrate Catalyze its chem transformation Often encloses substrate Substrate: mol bound to active site, acted upon enzyme ENZYMES AFFECT RXN RATES NOT EQ FUNC - increase rate or RXN Ground State (GS): starting point for either FOW or REV RXN Free ENG of sys by average mol under set of standard conditions (STP) Free ENG d = dG: standard free ENG chang Furthermore - bchm - dG' - the dG at STP but at 7 Ph EQ between S and P reflects the difference in free ENG of their ground states Position and direction of EQ are NOT AFFECTED by any catalyst FAV EQ does not mean S and P conversion will occur at detectable rate Rate of RXN DEP on ENG barrier (ENG req for alignment of reacting groups) Top of ENG = Transition State (TS): S and P equally probable (like a pKa of S and P) NOT CHM species with significant stability NOT RXN intermediate Temp moment in which: Bond breakage Bond formation Charge development
- proceed to precise point where decay of either S or P is EQUALLY likely
Enzymes are no exception to the rule that catalysts do not affect RXN EQ - resort FUNC: accelerate the interconversion btw S and P Enzyme is not used up and EQ is unaffected RXN reaches EQ faster because the rate is INC EX Sucrose + O2 --> CO2 + H2O [RXN very large and - dG' Sucrose is stable compound, must overcome (high aENG) before sucrose reacts with O2 Therefore sucrose can be stored with O2 almost indefinitely without reacting Sucrose in cells is readily broken down to CO2 and H2O DUE TO ENZYMES Reaction pathway is primary ENG yielding pathway of cells Enzymes of pathway allow RXN sequence to proceed in an biologically expedient manner Reaction Intermediates: chemical species that form and decay transiently ANY SPECIES that has a finite chemical lifetime S <--> P therefore ES and EP complexes are considered intermediates LOCATED AT VALLEYS IN RXN COORDINATE Even though S and P are stable chem species
Two fundamental qualifications of life Self replication Catalyze chem RXNs efficiently and effectively Enzymes central to every biochemical process Haldane made the remarkable suggestion that weak bonding interactions between an enzyme and its substrate might be used to catalyze a reaction What is an enzyme What is a cofactor What is a coenzyme What is a prosthetic group What is a holoenzyme What is a apoenzyme/apoprotein lysozyme was named for its ability to lyse bacterial cell walls The rate of a reaction reflects this activation energy: a higher activation energy corresponds to a slower reaction role of enzymes is to accelerate the interconversion of S and P A reaction intermediate is any species on the reaction pathway that has a finite chemical lifetime Without such energy barriers, complex macromolecules would revert spontaneously to much simpler molecular forms, and the complex and highly ordered structures and metabolic processes of cells could not exist.
A large negative value for _G__ reflects a favorable reaction equilibrium—but as already noted, this does not mean the reaction will proceed at a rapid rate.
First Order
Meaning that k = .03 s-1 that 3% is formed every 1 second Higher Keq means a higher affinity for the products Lower dG’ – standard free energy biochemical
Second Order
The important point here is that the relationship between the rate constant k and the activation energy _G‡ is inverse and exponential. In simplified terms, this is the basis for the statement that a lower activation energy means a faster reaction rate.
- Rate enhanced 5-17 orders of magnitude
- Enzymes are very specific
catalytic power of enzymes is derived from free energy released in forming many weak bonds and interactions between an enzyme and its substrate, binding energy contributes to specificity and catalysis weak interactions are optimized in RXN transition state, enzyme active sites are complementary not to the substrates but to transition states through which substrates pass as they are converted to products (during enzyme RXN)
How does an enzyme use binding energy to lower the activation energy for a reaction? Formation of the ES complex is not the explanation in itself, although some of the earliest considerations of enzyme mechanisms began with this idea Fischer and enzyme specificity 1894 enzymes were structurally complementary to their substrates – lock and key An enzyme completely complementary to its substrate would be a very poor enzyme, as we can demonstrate. EX Dihydrofolate reductase In reality, the complementarity between protein and ligand (in this case substrate) is rarely perfect, as we saw in Chapter 5. The interaction of a protein with a ligand often involves changes in the conformation of one or both molecules, a process called induced fit. This lack of perfect complementarity between enzyme and substrate (not evident in this figure) is important to enzymatic catalysis Polanyi and Haldane, elaborated by Pauling – in order to catalyze RXNs an enzyme must be complementary to the RXN transition state - optimal interactions between substrate and enzyme occur only in the transition state. To react (break), the stick must reach the transition state of the reaction, but the stick fits so tightly in the active site that it cannot bend, because bending would eliminate some of the magnetic interactions between stick and enzyme. Such an enzyme impedes the reaction, stabilizing the substrate instead. In a reaction coordinate diagram (Fig. 6–5b), this kind of ES complex would correspond to an energy trough from which the substrate would have difficulty escaping. Such an enzyme would be useless.
Analogous enzyme RXN Now, however, the increase in free energy required to draw the stick into a bent and partially broken conformation is offset, or “paid for,” by the magnetic interactions (binding energy) that form between the enzyme and substrate in the transition state. Many of these interactions involve parts of the stick that are distant from the point of breakage; thus interactions between the stickase and nonreacting parts of the stick provide some of the energy needed to catalyze stick breakage This “energy payment” translates into a lower net activation energy and a faster reaction rate
Real Enzymes Weak interactyion are formed in the ES complex, full complement of interactions between S and ENZ is formed only when the S reaches the transition state Free energy released by the formation of these interactions partially offsets the ENG require dto reac he top of the ENG hill POS dG (dcross) and NEG bENG DEC net aENG Transition states is not a stable speices but a brief point in time that the substrate spends atop the ENG hill The enzyme catalyzed reactions is much faster because the hills is much smaller
Consider an imaginary reaction, the breaking of a magnetized metal stick. The uncatalyzed reaction is shown in Figure 6–5a. Let’s examine two imaginary enzymes—two “stickases”—that could catalyze this reaction, both of which employ magnetic forces as a paradigm for the binding energy used by real enzymes. We first design an enzyme perfectly complementary to the substrate (Fig. 6–5b). The active site of this stickase is a pocket lined with magnets. To react (break), the stick must reach the transition state of the reaction, but the stick fits so tightly in the active site that it cannot bend, because bending would eliminate some of the magnetic interactions between stick and enzyme. Such an enzyme impedes the reaction, stabilizing the substrate instead. In a reaction coordinate diagram (Fig. 6–5b), this kind of ES complex would correspond to an energy trough from which the substrate would have difficulty escaping. Such an enzyme would be useless. The modern notion of enzymatic catalysis, first proposed by Michael Polanyi (1921) and Haldane (1930), was elaborated by Linus Pauling in 1946: in order to catalyze reactions, an enzyme must be complementary to the reaction transition state. This means that optimal interactions between substrate and enzyme occur only in the transition state. Figure 6–5c demonstrates how such an enzyme can work. The metal stick binds to the stickase, but only a subset of the possible magnetic interactions are used in forming the ES complex. The bound substrate must still undergo the increase in free energy needed to reach the transition state. Now, however, the increase in free energy required to draw the stick into a bent and partially broken conformation is offset, or “paid for,” by the magnetic interactions (binding energy) that form between the enzyme and substrate in the transition state. Many of these interactions involve parts of the stick that are distant from the point of breakage; thus interactions between the stickase and nonreacting parts of the stick provide some of the energy needed to catalyze stick breakage. This “energy payment” translates into a lower net activation energy and a faster reaction rate. Real enzymes work on an analogous principle. Some weak interactions are formed in the ES complex, but the full complement of such interactions between substrate and enzyme is formed only when the substrate reaches the transition state. The free energy (binding energy) released by the formation of these interactions partially offsets the energy required to reach the top of the energy hill. Even on the enzyme, the transition state is not a stable species but a brief point in time that the substrate spends atop an energy hill. The enzymecatalyzed reaction is much faster than the uncatalyzed process, however, because the hill is much smaller. The summation of the unfavorable POS aENG and NEG bENG lower net aENG When the enzyme is complementary to the substrate (b), the ES complex is more stable and has less free energy in the ground state than substrate alone. The result is an increase in the activation energy. weak binding interactions between the enzyme and the substrate provide a substantial driving force for enzymatic catalysis.
The requirement for multiple weak interactions to drive catalysis is one reason why enzymes (and some coenzymes) are so large. An enzyme must provide functional groups for ionic, hydrogen-bond, and other interactions, and also must precisely position these groups so that binding energy is optimized in the transition state. Adequate binding is accomplished most readily by positioning a substrate in a cavity (the active site) where it is effectively removed from water. Size reflects the need for the superstructure to keep interacting and to prevent the cavity from collapsing
The same binding energy that provide ENG for catalysis also gives an enzyme its specificity – to discriminate between a substrate and a competing molecule Catalysis and specificity arise from the same phenomenon If an enzyme active site has functional groups arranged optimally to form a variety of weak interactions with a particular substrate in the transition state, the enzyme will not be able to interact to the same degree with any other molecule specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate molecule. binding energy makes an important, and sometimes the dominant, contribution to catalysis. Consider what needs to occur for a reaction to take place. These mechanisms are not mutually exclusive, and a given enzyme might incorporate several types in its overall mechanism of action. For most enzymes, it is difficult to quantify the contribution of any one catalytic mechanism to the rate and/or specificity of a particular enzyme-catalyzed reaction.
might include (1) a reduction in entropy, in the form of decreased freedom of motion of two molecules in solution; (2) the solvation shell of hydrogen-bonded water that surrounds and helps to stabilize most biomolecules in aqueous solution; (3) the distortion of substrates that must occur in many reactions; and (4) the need for proper alignment of catalytic functional groups on the enzyme.
First, a large restriction in the relative motions of two substrates that are to react, or entropy reduction, is one obvious benefit of binding them to an enzyme Binding energy holds the substrates in the proper orientation to react—a substantial contribution to catalysis, because productive collisions between molecules in solution can be exceedingly rare Substrates can be precisely aligned on the enzyme, with many weak interactions between each substrate and strategically located groups on the enzyme clamping the substrate molecules into the proper positions. Studies have shown that constraining the motion of two reactants can produce rate enhancements of many orders of magnitude Second, formation of weak bonds between substrate and enzyme also results in desolvation of the substrate. Enzyme-substrate interactions replace most or all of the hydrogen bonds between the substrate and water. Third, binding energy involving weak interactions formed only in the reaction transition state helps to compensate thermodynamically for any distortion, primarily electron redistribution, that the substrate must undergo to react. Finally, the enzyme itself usually undergoes a change in conformation when the substrate binds, induced by multiple weak interactions with the substrate
Note that the reactant in (b) has freedom of rotation about three bonds (shown with curved arrows), but this still represents a substantial reduction of entropy over (a). If the bonds that rotate in (b) are constrained as in (c), the entropy is reduced further and the reaction exhibits a rate enhancement of 108 M relative to (a). This is referred to as induced fit, a mechanism postulated by Daniel Koshland in 1958 serves to bring specific functional groups on the enzyme into the proper position to catalyze the reaction. The conformational change also permits formation of additional weak bonding interactions in the transition state new enzyme conformation has enhanced catalytic properties binding energy used to form the ES complex is just one of several contributors overall catalytic mechanism Once a substrate is bound to an enzyme, properly positioned catalytic functional groups aid in the cleavage and formation of bonds by a variety of mechanisms, including general acid-base catalysis, covalent catalysis, and metal ion catalysis distinct from mechanisms based on binding energy, because they generally involve transient covalent interaction with a substrate or group transfer to or from a substrate Catalysis of this type that uses only the H_ (H3O_) or OH_ ions present in water is referred to as specific acid-base catalysis If protons are transferred between the intermediate and water faster than the intermediate breaks down to reactants, the intermediate is effectively stabilized every time it forms. No additional catalysis mediated by other proton acceptors or donors will occur. general acid-base catalysis refers to proton transfers mediated by other classes of molecules nonenzymatic reactions in aqueous solutions, this occurs only when the unstable reaction intermediate breaks down to reactants faster than protons can be transferred to or from water Many weak organic acids can supplement water as proton donors in this situation, or weak organic bases can serve as proton acceptors.
Covalent Catalysis transient covalent bond is formed between the enzyme and the substrate. Consider the hydrolysis of a bond between groups A and B:
This alters the pathway of the reaction, and it results in catalysis only when the new pathway has a lower activation energy than the uncatalyzed pathway Both of the new steps must be faster than the uncatalyzed A number of amino acid side chains and the functional groups of some enzyme cofactors can serve as nucleophiles in the formation of covalent bonds with substrates These covalent complexes always undergo further reaction to regenerate the free enzyme. The covalent bond formed between the enzyme and the substrate can activate a substrate for further reaction in a manner that is usually specific to the particular group or coenzyme. Metals, whether tightly bound to the enzyme or taken up from solution along with the substrate, can participate in catalysis in several ways. Ionic interactions between an enzyme-bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states. Metals can also mediate oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state
Most enzymes employ a combination of several catalytic strategies to bring about a rate enhancement. A good example of the use of both covalent catalysis and general acid-base catalysis is the reaction catalyzed by EX chymotrypsin.
E enhance reaction rates by lowering aENG E catalyze thermodynamically favorable RNXs Catalysts ONLY affect reaction rates
Three dimensional structure of the protein provides important informatoin and site directed mutagenesis mutagenesis - changing amino acid sequences of a protein by genetic engineering Enzyme Kinetics: Central approach to studying the mechanism of an enzyme catalyzed RXN and how it changes in response to changes in experimental parameters oldest but most important at analyzing enzymes
[S] key factor that affects the Rate of Enzyme Catalyzed RXNs Complicated by the [S] changes during the course of an in vitro RXN as S --> P One approach Measure the initial rate/velocity (Vo) when [S] > [E] where [S] is in 5-6 orders in magnitide higher than [E] Beginning - changes in [S] can be limited to a few % and [S] regarded as constant Vo can be explored as funciton of [S] - when adjusted by investigator @ low [S], Vo INC linearly with INC in [S] @ high [S], Vo INC smaller with INC in [S] Maximal Velocity (Vmax): point where INC in Vo INC negligibly small with INC in [S] Another approach ES complex is the key to understanding this kinetic behavior (Henri - combination of an E with its S to form ES complex is a necessary step in enzyme catalysis) (Michaelis & Menten - E first combines w/ S to form ES complex in a fast reverisble step) E+S <-(k-1)- -(k1)-> ES - Eqn1 (ES complex breaks down in slower second step --> free E and P) ES <-(k-2)- -(k2)-> E + P - Eqn2 (slower second RXN limits the rate of overall RXN) (Overall rate must be proportional to [ES] or the species that reacts in second step)
@ any given instant enzyme catalyzed RXN exists in two forms: (1f) Free or uncombined E (2f) Combined ES @ low [S] most of enzyme is in (1f) rate is proportional to [S] WHY? EQ of Eqn1 is pushed toward formation of more ES as [S] INC
