8885dc06190-2371/27/047:13 AM Page199mac76mac76:385 6.2 How Enzymes Work This reaction rearranges the carbonyl and hydroxyl groups on carbons 1 and 2. However, more than 80% of the enzymatic rate acceleration has been traced to uncat enzyme-substrate interactions involving the phosphate group on carbon 3 of the substrate. This was determined by a careful comparison of the enzyme-catalyzed reactions with glyceraldehyde 3-phosphate and with glyceraldehyde(no phosphate group at position 3)as substrate Reaction coordinate The general principles outlined above can be illus- trated by a variety of recognized catalytic mechanisms FIGURE 6-6 Role of binding energy in catalysis. To lower the acti. These mechanisms are not mutually exclusive, and a ation energy for a reaction, the system must acquire an amount of given enzyme might incorporate several types in its energy equivalent to the amount by which AG is lowered. Much of overall mechanism of action. For most enzymes, it is dif- this energy comes from binding energy (ACB)contributed by forma- ficult to quantify the contribution of any one catalytic tion of weak noncovalent interactions between substrate and enzyme mechanism to the rate and/or specificity of a particular in the transition state. The role of AGg is analogous to that of AGm in enzyme-catalyzed reaction Figure 6-5 As we have noted, binding energy makes an impor tant and sometimes the dominant contribution to catal- rsis. Consider what needs to occur for a reaction to take ergies by the 60 to 100 kJ/mol required to explain the place. Prominent physical and thermodynamic factors large rate enhancements observed for many enzymes. contributing to AG, the barrier to reaction, might in- The same binding energy that provides energy for clude (1)a reduction in entropy, in the form of de catalysis also gives an enzyme its specificity, the abil- creased freedom of motion of two molecules in solution ity to discriminate between a substrate and a competing (2) the solvation shell of hydrogen-bonded water that molecule. Conceptually, specificity is easy to distinguish surrounds and helps to stabilize most biomolecules in from catalysis, but this distinction is much more difficult aqueous solution; ( 3) the distortion of substrates that to make experimentally, because catalysis and specificity must occur in many reactions; and(4) the need for arise from the same phenomenon. If an enzyme active proper alignment of catalytic functional groups on the site has functional groups arranged optimally to form a enzyme. Binding energy can be used to overcome all variety of weak interactions with a particular substrate these barriers in the transition state, the enzyme will not be able to in- First, a large restriction in the relative motions of teract to the same degree with any other molecule. For wo substrates that are to react, or entropy reduction, example, if the substrate has a hydroxyl group that forms is one obvious benefit of binding them to an enzyme. a hydrogen bond with a specific Glu residue on the en- Binding energy holds the substrates in the proper ori zyme, any molecule lacking a hydroxyl group at that par- entation to react-a substantial contribution to cataly- ticular position will be a poorer substrate for the enzyme. sis, because productive collisions between molecules il ddition, any molecule with an extra functional group solution can be exceedingly rare. Substrates can be pre- for which the enzyme has no pocket or binding site is cisely aligned on the enzyme, with many weak interac likely to be excluded from the enzyme. In general, spec tions between each substrate and strategically located ficity is derived from the formation of many weak in- groups on the enzyme clamping the substrate molecules teractions between the enzyme and its specific substrate into the proper positions. Studies have shown that con molecule straining the motion of two reactants can produce rate The importance of binding energy to catalysis can enhancements of many orders of magnitude(Fig. 6-7 e readily demonstrated. For example, the glycolyti Second formation of weak bonds between substrate enzyme triose phosphate isomerase catalyzes the inter- and enzyme also results in desolvation of the substrate conversion of glyceraldehyde 3-phosphate and dihy Enzyme-substrate interactions replace most or all of the dioxyacetone phosphate hydrogen bonds between the substrate and water Third, binding energy involving weak interactions formed only in the reaction transition state helps to HC-OH compensate thermodynamically for any distortion, pri- HC-OH trios marily electron redistribution, that the substrate must CH2OPo3- phosphate CH,OPo32 undergo to react Finally, the enzyme itself usually undergoes a hange in conformation when the substrate binds, in- duced by multiple weak interactions with the substrate.ergies by the 60 to 100 kJ/mol required to explain the large rate enhancements observed for many enzymes. The same binding energy that provides energy for catalysis also gives an enzyme its specificity, the abil￾ity to discriminate between a substrate and a competing molecule. Conceptually, specificity is easy to distinguish from catalysis, but this distinction is much more difficult to make experimentally, because 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 in￾teract to the same degree with any other molecule. For example, if the substrate has a hydroxyl group that forms a hydrogen bond with a specific Glu residue on the en￾zyme, any molecule lacking a hydroxyl group at that par￾ticular position will be a poorer substrate for the enzyme. In addition, any molecule with an extra functional group for which the enzyme has no pocket or binding site is likely to be excluded from the enzyme. In general, speci￾ficity is derived from the formation of many weak in￾teractions between the enzyme and its specific substrate molecule. The importance of binding energy to catalysis can be readily demonstrated. For example, the glycolytic enzyme triose phosphate isomerase catalyzes the inter￾conversion of glyceraldehyde 3-phosphate and dihy￾droxyacetone phosphate: This reaction rearranges the carbonyl and hydroxyl groups on carbons 1 and 2. However, more than 80% of the enzymatic rate acceleration has been traced to enzyme-substrate interactions involving the phosphate group on carbon 3 of the substrate. This was determined by a careful comparison of the enzyme-catalyzed reactions with glyceraldehyde 3-phosphate and with glyceraldehyde (no phosphate group at position 3) as substrate. The general principles outlined above can be illus￾trated by a variety of recognized catalytic mechanisms. 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 dif￾ficult to quantify the contribution of any one catalytic mechanism to the rate and/or specificity of a particular enzyme-catalyzed reaction. As we have noted, binding energy makes an impor￾tant, and sometimes the dominant, contribution to catal￾ysis. Consider what needs to occur for a reaction to take place. Prominent physical and thermodynamic factors contributing to G‡ , the barrier to reaction, might in￾clude (1) a reduction in entropy, in the form of de￾creased 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. Binding energy can be used to overcome all these barriers. 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 ori￾entation to react—a substantial contribution to cataly￾sis, because productive collisions between molecules in solution can be exceedingly rare. Substrates can be pre￾cisely aligned on the enzyme, with many weak interac￾tions between each substrate and strategically located groups on the enzyme clamping the substrate molecules into the proper positions. Studies have shown that con￾straining the motion of two reactants can produce rate enhancements of many orders of magnitude (Fig. 6–7). 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, pri￾marily electron redistribution, that the substrate must undergo to react. Finally, the enzyme itself usually undergoes a change in conformation when the substrate binds, in￾duced by multiple weak interactions with the substrate. 6.2 How Enzymes Work 199 ‡ Reaction coordinate S P G‡ uncat G‡ cat ‡ ES EP GB Free energy, G FIGURE 6–6 Role of binding energy in catalysis. To lower the acti￾vation energy for a reaction, the system must acquire an amount of energy equivalent to the amount by which G‡ is lowered. Much of this energy comes from binding energy (GB) contributed by forma￾tion of weak noncovalent interactions between substrate and enzyme in the transition state. The role of GB is analogous to that of GM in Figure 6–5. triose phosphate isomerase Glyceraldehyde 3-phosphate HC CH2OPO3 2 CH2OPO3 2 H2C C 1 HC OH 2 3 O Dihydroxyacetone phosphate OH O 8885d_c06_190-237 1/27/04 7:13 AM Page 199 mac76 mac76:385_reb: