Role of Induced Fit in Enzyme Specificity: A Molecular Forward/Reverse Switch
Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product. Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product. The role of induced fit in enzyme specificity has been controversial. On the one hand, it is apparent that catalysis is facilitated by the rapid binding of a substrate to an open form of the enzyme, whereas the chemical reaction is accelerated most efficiently by the precise alignment of amino acids surrounding the substrate and by the altered reaction environment in the closed state. At this level, it is apparent that changes in enzyme structure may simply solve the disparate demands for an open site to allow fast binding and a closed site to afford fast catalysis. It has been argued that one- and two-step binding reactions would lead to the same end point with the same free energy change, and therefore, induced fit can do nothing to improve catalytic efficiency or specificity (1Fersht A.R. Enzyme Structure and Mechanism. 3rd Ed. W. H. Freeman, New York1999Google Scholar). However, these arguments fail to consider the rates at which the enzyme closes and opens following the initial substrate binding. A general mechanism, including isomerization after an initial weak substrate binding, can be described by the following reaction sequence (Scheme 1). E+S⇄K1ES⇄k2FS⇄k3FP⇄k4E+PSCHEME 1 The substrate first binds to the open form of the enzyme (E) and then induces a change in structure to the closed form (FS). The relative values for the forward and reverse rates of the conformational change (k2 and k–2) and the rate of the chemical reaction (k3) provide the key to understanding the role of the conformational change in enzyme specificity. Surprisingly, the rate of the reverse conformational change (k–2) is an important specificity determinant (2Tsai Y.C. Johnson K.A. Biochemistry. 2006; 45: 9675-9687Crossref PubMed Scopus (206) Google Scholar). The term induced fit was first proposed to explain why hexokinase had such low ATPase activity. The simple logic said that when ATP bound in the absence of glucose, the ATP must be protected from water, whereas the binding of glucose must induce a change in structure to make the ATP accessible (3Koshland D.E. Boyer P.D. Lardy H. Myrback K. The Enzymes. 2nd Ed. 1. Academic Press, New York1959: 305-346Google Scholar). This basic concept was confirmed by the solution of the crystal structure of hexokinase (4Bennett Jr., W.S. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4848-4852Crossref PubMed Scopus (279) Google Scholar). Decades later, the solution of the structure of the ribosome has revealed a “steric switch mechanism” that protects the peptidyl-tRNA from hydrolysis, whereas the binding of a cognate aa-tRNA 2The abbreviations used are: aa-tRNAaminoacyl-tRNAEF-Tuelongation factor TudNTPdeoxynucleoside triphosphateDHFRdihydrofolate reductase. induces a change in structure to allow reaction (5Schmeing T.M. Huang K.S. Strobel S.A. Steitz T.A. Nature. 2005; 438: 520-524Crossref PubMed Scopus (283) Google Scholar). Steps involved in delivering the aa-tRNA to the ribosome are complex, involving EF-Tu and the hydrolysis of GTP, but nonetheless reveal some common underlying themes (6Rodnina M.V. Gromadski K.B. Kothe U. Wieden H.J. FEBS Lett. 2005; 579: 938-942Crossref PubMed Scopus (120) Google Scholar). aminoacyl-tRNA elongation factor Tu deoxynucleoside triphosphate dihydrofolate reductase. Structures provide clues as to how a desired reaction can be favored by the alignment of catalytic residues. However, the manner in which changes in enzyme structure influence specificity remains obscure, bearing in mind that enzyme specificity is a purely kinetic property with less than obvious structural origins. DNA polymerases offer a unique model for accessing specificity because the alternate substrates are well defined, and the discrimination between correct and incorrect substrates can be quite high. Analysis of DNA polymerases has led to the suggestion that the binding of the correct base (as defined by base pairing with the template strand) induces a change in structure to facilitate catalysis, whereas the binding of a mismatched base may somehow fail to induce the same organization of catalytic residues (7Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (507) Google Scholar, 8Xiang Y. Goodman M.F. Beard W.A. Wilson S.H. Warshel A. Proteins. 2008; 70: 231-247Crossref PubMed Scopus (43) Google Scholar). Early work on DNA polymerases focused on deducing whether the conformational change may limit and thereby regulate the rate of incorporation (7Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (507) Google Scholar). Subsequent structural studies demonstrated a large change in the “fingers” domain after binding the correct dNTP, supporting the notion of an induced fit (9Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1105) Google Scholar). Models were developed suggesting that each step along the pathway (ground state binding, conformational change, and chemistry) contributed a checkpoint such that the net fidelity was the product of the contributions at each step (10Joyce C.M. Benkovic S.J. Biochemistry. 2004; 43: 14317-14324Crossref PubMed Scopus (281) Google Scholar), and arguments focused on whether the conformational change or the chemistry step was rate-limiting (11Showalter A.K. Tsai M.D. Biochemistry. 2002; 41: 10571-10576Crossref PubMed Scopus (128) Google Scholar, 12Rothwell P.J. Mitaksov V. Waksman G. Mol. Cell. 2005; 19: 345-355Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We now know that for the past two decades we have been asking the wrong question. To define the role of the conformational change in enzyme specificity, we need to compare the rate of the reverse of the conformational change with the rate of the chemistry step, not the rate of the forward conformational change step. The analysis leading to this unexpected conclusion is summarized below. To examine the kinetics of the conformational change, a fluorescent label was placed on the fingers domain of T7 DNA polymerase at a position that would be sensitive to changes in protein structure in forming the closed complex (2Tsai Y.C. Johnson K.A. Biochemistry. 2006; 45: 9675-9687Crossref PubMed Scopus (206) Google Scholar). This allowed the rate and equilibrium constants governing substrate binding and the conformational change to be measured, and the results were combined with conventional measurement of the rate of the chemistry step by rapid quench-flow methods using radiolabeled DNA. These studies led to the following pathway for correct nucleotide binding and incorporation (Scheme 2), E·Dn+N⇄28μME·Dn·NSCHEME 2 E·Dn·N⇄1.6s-1660s-1F·Dn·N⇄360s-1E·Dn+1+PP where E·Dn represents the enzyme·DNA complex with a primer strand n residues in length, N represents the incoming dNTP, and F·Dn·N represents the closed state of the enzyme. The rates of substrate binding and release (k1 and k–1) are not known but can be approximated based upon conservative estimates of diffusion-controlled binding (k1 ≥ 100 μm–1 s–1) and the measured dissociation constant of 28 μm (k–1 ≥ 2800 s–1). Pyrophosphate release appears to be fast, so this simple three-step mechanism is sufficient to account for processive synthesis (13Hanes J.W. Johnson K.A. Nucleic Acids Res. 2007; 35: 6973-6983Crossref PubMed Scopus (38) Google Scholar). Steady-state analysis of two competing substrates (or two competing enzymes) shows that specificity is a linear function of kcat/Km. Therefore, to understand the role of conformational changes, we need only to define the effect of the substrate-induced conformational changes on the value of kcat/Km. The simple math is reproduced here because the results are quite compelling. The specificity constant for this pathway is defined by Equation 1. kcat/Km=k1k2k3k2k3+k-1(k-2+k3)(Eq. 1) Because the rate of chemistry (k3) is much greater than the reverse of the conformational change step (k–2), this reduces to Equation 2. kcat/Km≈k1k2k3k2k3+k-1k3=k1k2k2+k-1(Eq. 1) This leads to the surprising conclusion that the rate of chemistry does not enter into the definition of kcat/Km even though it is slower than the conformational change step! This is because the reverse of the conformational change step is so slow relative to chemistry that the substrate is committed to go forward after the conformational change. The equation defining kcat/Km can be further reduced based upon the rapid equilibrium binding of substrate in the collision complex by the comparison indicating that k–1 ≫ k2 to yield Equation 3. kcat/Km≈K1k2(Eq. 3) Thus, kcat/Km is defined by the product of the binding constant (K1) for the substrate in the collision complex (ES) and the rate of the conformational change (k2). It is important to note, however, that kcat is not equal to k2 and Km is not equal to 1/K1; additional terms in both kcat and Km cancel in the ratio kcat/Km. Although it is often taught that kcat/Km is the ratio of kcat and Km, this view often leads to inaccurate conclusions, especially when the assumption is made that kcat measures the rate of chemistry and Km is equal to the ground state dissociation constant for substrate (1Fersht A.R. Enzyme Structure and Mechanism. 3rd Ed. W. H. Freeman, New York1999Google Scholar). Rather, the specificity constant is best understood as the second-order rate constant for substrate binding times the probability that, once bound, the substrate continues forward to form product. According to our model for correct dNTP incorporation, the second-order rate constant for substrate binding is K1k2, and the probability that the bound substrate continues forward is near unity. This result also brings forth the cautionary note that analysis of the separate contributions of kcat and Km to discrimination can be misleading (14Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (619) Google Scholar). As seen here, kcat contains terms (notably k3) that do not contribute to the specificity constant for the correct base. Analysis of the kinetics of the conformational change following the binding of a mismatch yielded more surprises. First, the binding of a mismatch caused an increase in fluorescence, opposite to that seen following the binding of a correct base. This implies that there are three distinct structural states, open (defined by the structure in the absence of bound nucleotide), closed (defined by the structure seen in the ternary enzyme·DNA·dNTP complex), and a unique mismatch recognition state that has not yet been solved crystallographically. Moreover, the kinetics of the fluorescence change after mismatch binding were suggesting conformational with equilibrium The data were to a model (Scheme constants governing are quite from leading to incorporation of a correct base. The reverse rate of the conformational change is greater than the forward so the isomerization is and the rate of the chemical reaction is reduced from that seen with the correct substrate. In this of the equation for the specificity constant Equation The rate of incorporation is so slow relative to the reverse of the conformational change step that the leading to incorporation, and therefore, the specificity constant is the product of the two equilibrium constants and and the rate of the chemistry step. The most important conclusion of this analysis is that binding of a mismatched nucleotide leads to a state from which dissociation of the bound nucleotide is favored the incorporation rate is The data that the enzyme has to recognize a mismatch and to binding energy to catalytic residues and of the binding state seen with a correct substrate. Thus, the conformational change acts as a switch to recognize a correct substrate and it catalytic residues to recognize an incorrect substrate and dissociation catalytic residues. The fidelity of T7 DNA polymerase is to the kinetic of the conformational after nucleotide binding. The kinetic of the conformational is defined by A correct base induces a state in which of the bound substrates in the forward whereas a mismatch induces a state in which only 1). The kinetic of the conformational is also in the free energy in The show how the correct substrate to the forward reaction the bound mismatch release to the relative of the of conformational for three and initial binding of the aa-tRNA EF-Tu complex to the ribosome is not a rapid so kcat/Km dihydrofolate and The initial binding of the aa-tRNA EF-Tu complex to the ribosome is not a rapid so kcat/Km A. M.V. W. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google dihydrofolate and in a It has been argued that fidelity of a polymerase is a function of kinetic the first ground state binding, the the conformational change step, and the to the rate of chemistry (10Joyce C.M. Benkovic S.J. Biochemistry. 2004; 43: 14317-14324Crossref PubMed Scopus (281) Google Scholar). The that the net is simply the product of the values at the three is not at for the fidelity T7 DNA In this the net discrimination is the ratio of kcat/Km values for correct and incorrect substrates and is by Equation In this only the ratio of the ground state binding constants (K1) for correct and incorrect substrates into the value for the net It remains to be whether DNA polymerases this same as of this studies have been in which the reverse rate (k–2) has been measured for of the the past two we and have been on whether the conformational change or chemistry was rate-limiting k2 and However, we have been the rate of the reverse of the change with the rate of the chemical reaction and In these kinetic to one is with the of how the rate of the conformational change can be by the structure of the enzyme and the of the with the bound substrate. the that we as must be the of to the ground state binding leading to a of enzyme that a state are made to recognize the correct substrate and the change in may be structural changes to bring catalytic residues into precise alignment for catalysis, and work has that at bring the to the alignment to the state Y. Goodman M.F. Beard W.A. Wilson S.H. Warshel A. Proteins. 2008; 70: 231-247Crossref PubMed Scopus (43) Google Scholar, S.J. S. T. P.J. S. Biochemistry. 2008; PubMed Scopus Google Scholar). These would be a of the net rate constant for the chemistry step As kcat is an important especially for a DNA polymerase where the demands be by more enzyme. However, once a is where is greater than further in do not improve only increase We proposed that the correct and incorrect substrates induce structural based upon the that the binding of a mismatch induced a change in fluorescence opposite in to that after binding a correct base (2Tsai Y.C. Johnson K.A. Biochemistry. 2006; 45: 9675-9687Crossref PubMed Scopus (206) Google Scholar). incorrect base at a rate slower than a correct base even though the chemical reaction for correct and incorrect base are Therefore, these data that the binding of a mismatch leads to of catalytic suggesting a free energy in In this the free energy at each of the and at each point is defined by our but the of the structural is have proposed a free energy but these upon the the point in the state from the bound substrate to product Y. Goodman M.F. Beard W.A. Wilson S.H. Warshel A. Proteins. 2008; 70: 231-247Crossref PubMed Scopus (43) Google Scholar, S.J. S. T. P.J. S. Biochemistry. 2008; PubMed Scopus Google and the of the but do not the kinetic of the conformational as it to specificity. analysis has focused on the Annu. Rev. Biochem. 2002; PubMed Scopus Google of enzyme catalysis by to how the enzyme the state and the chemical reaction at the site Y. Goodman M.F. Beard W.A. Wilson S.H. Warshel A. Proteins. 2008; 70: 231-247Crossref PubMed Scopus (43) Google Scholar, S.J. S. T. P.J. S. Biochemistry. 2008; PubMed Scopus Google Scholar). an even would be to how the weak binding of the substrate can then a large conformational change leading to binding and the alignment of catalytic residues. may be to such and slow 2005; PubMed Scopus Google Scholar). Analysis of enzyme has led to the of enzyme into catalytic and specificity Annu. Rev. Biochem. 70: PubMed Scopus Google Scholar). Although this is from a purely structural and it is only when residues in the specificity domain and the in with the catalytic that such binding can be into catalytic efficiency and specificity. it is that the specificity specificity by binding the substrates thereby to the forward reaction. of the cognate aa-tRNA protein synthesis is also by the of the substrate dissociation rate for the correct substrate A. M.V. W. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). The in a complex with binds to the recognition leads to of hydrolysis, which is the rate-limiting step leading to of the aa-tRNA into the of the ribosome and rapid and aa-tRNA are by the forward reverse of an after a conformational change 1). a cognate of the forward forward whereas for a forward Although the reaction sequence is complex with where changes in are for rapid incorporation, it is to note that the point of discrimination the kinetic of the conformational after the initial substrate binding. has been to conformational changes upon substrate binding, but of these to be Johnson K.A. Benkovic S.J. Biochemistry. PubMed Scopus Google Scholar). these one can understand the second-order rate constant for substrate binding as the product of a weak binding constant for the initial collision complex and the rate of (Scheme 1). the kinetic of the conformational is defined by the relative values for and 1). the substrate the substrate release rate is whereas the rate of the chemistry step is the relative of and the specificity constant for dihydrofolate is determined solely by the rate of binding, μm–1 Although the kinetics of an incorrect substrate have not been defined, one can examine the rates for the product of the as a reaction defined in the forward with as the whereas so the kinetic favors release rather than the to do chemistry or release the bound substrate appears to be made the conformational change. Recent studies on have focused on on a and to be for catalysis Benkovic S.J. H.J. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). Moreover, it has been that the or after the chemical reaction bring the enzyme from a with the substrates bound, to a with the bound S. Benkovic S.J. T. P.J. S. Annu. Rev. Biochem. 2006; 75: PubMed Scopus Google Scholar). This structural to or following catalysis, also changes the kinetic from reaction of the bound substrate to release of the bound product. It is to note that may have not only to the conformational changes to favor the binding and alignment of the desired but also to conformational changes to the release rather than reverse reaction of the product of the reaction. The initial of the concept of an induced fit was that an reaction can be whereas the desired reaction can be facilitated by structural upon substrate binding. this it is now apparent that the role of conformational changes in alignment of catalytic residues and the environment at the site to catalysis to be Moreover, it now appears that the may also be in suggesting that the of catalytic residues be used to slow the reaction of an substrate (or product of the arguments the of an induced fit mechanism to alter specificity have each been by that, in to be (1Fersht A.R. Enzyme Structure and Mechanism. 3rd Ed. W. H. Freeman, New York1999Google argued an induced fit mechanism for specificity based upon the assumption that the isomerization reaction was fast and not rate-limiting and that the structure of the closed state was for The pathway does not we consider only equilibrium end and therefore, it is obvious that there is between with or without a conformational change In this the assumption of rapid and the conclusion that the pathway does not are Scopus Google argued on that the conformational change would need to be rate-limiting to specificity, and our work (7Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (507) Google on DNA polymerases that this was the and Jr., Biochemistry. PubMed Scopus Google the concept that the structure of the complex in the state may between correct and incorrect for a specificity to the induced fit even the conformational change was of a rapid equilibrium binding. most data that the conformational change is not rate-limiting but specificity and that correct and incorrect substrates induce conformational In the described here, the chemistry step is ≫ or the product release is fast ≫ so the rates of and do not influence specificity, can in the most general it has been that the DNA polymerase has a mechanism of discrimination and release is the chemical reaction to to equilibrium at the site (13Hanes J.W. Johnson K.A. Nucleic Acids Res. 2007; 35: 6973-6983Crossref PubMed Scopus (38) Google Scholar, J.W. Johnson K.A. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The reverse reaction the of the complex, thereby the probability of substrate release and kcat/Km. an important here is that, in the most general specificity can be by step in the and need to be measured to an In we have now the of the substrate dissociation rate in governing so the conformational change step need not be rate-limiting to the specificity even chemistry is rate-limiting in the it have influence on specificity as as the rate of chemistry is greater than the rate at which the substrate In we have that the conformational after the binding of a correct an incorrect substrate (2Tsai Y.C. Johnson K.A. Biochemistry. 2006; 45: 9675-9687Crossref PubMed Scopus (206) Google Scholar). In the based upon are to be Rather, it appears as though have to of to improve specificity and efficiency at substrate A substrate can be favored by binding in the collision complex, by a rate of the conformational change, and by a closed state with a substrate release rate and an alignment of catalytic residues. In and (Scheme 1) product In contrast, an incorrect substrate may be by binding to the collision complex and isomerization to a state that rapid release catalytic residues. Moreover, and can also kcat/Km. is for that an enzyme is somehow from of of these structural and kinetic to improve specificity. The isomerization following substrate binding appears to as a molecular switch by whether to on to a substrate and residues to catalysis or to favor release of an substrate the rate of reaction. of a and on a may then further facilitate the chemical reaction. The of is an important leading to specificity and