Dynamic Model for Enzyme Action

Protein & Peptide Letters, 2011, 18, 000-000

1

Dynamic Model for Enzyme Action Qinyi Zhao* Dept. of Biochemistry and Molecular Biology, Medical Institute, P. O. Box 2619, Beijing 100068, PR China Abstract: Introduction: Protein thermodynamic structure theory is an integrated approach to the study of protein dynamics and the mechanisms of enzyme catalysis. In this paper, a hypothesis arising from this theory is examined. Hypothesis: The timescale of an enzymatic reaction (TER) gives a key to characterizing enzyme conformational changes. The aspects of timescale important in our approach are: (i) it is logically related to internal motions of the main chain of a protein; (ii) it sets the upper limit on the size or scope of protein conformational changes. Feature (i) is linked to the dynamic properties of enzyme-reactant complexes. Feature (ii) is linked to the dynamic sites of the main chain (promoting motion) involved in enzyme activity. Conclusion: Our analysis shows that a comprehensive understanding of enzymology can be established on the basis of protein thermodynamic structure theory.

Keywords: Motion, enzyme, flexibility, mechanism, conformation, dynamics INTRODUCTION Interpreting the working mechanism of enzyme action is a fundamental task of enzymology. The traditional theory of enzyme action was established on the basis of protein threedimensional structure and transition state theory (1). Remarkable achievements have been made, but the relationship between enzyme activity and conformational changes in enzymes has not been well established theoretically. In order to give a reasonable explanation for the high efficiency of enzyme activity, Pauling proposed that the enzyme binds tightly to the transition state of the substrate (2). The “induced-fit” hypothesis, proposed by Koshland (3, 4), fundamentally changed our thought about enzymology. This model proposes that enzymes have rather flexible structures and the interaction between enzyme and substrate rapidly induces reshaping of the enzyme structure at the active site, thus strengthening the binding between enzyme and substrate. The concept of protein flexibility, applied to various features of proteins such as structure, function and dynamic properties, therefore becomes an important topic in enzymology as well as protein science. Some scientists have further concluded that catalytic activity results from the flexibility (or rapid internal motions) of the active site of an enzyme (5-7). The “energetic pre-organization of active sites” hypothesis was proposed by Warshel. This hypothesis states that the enzymic environment is complementary to the transition state configuration of the reactants, so it greatly reduces the activation energy of an enzyme-catalyzed reaction (8, 9). Another approach to enhance the understanding of protein science was protein dynamics (10) and the conformation selection mechanism for ligand-protein binding (11, 12, 13). According to this perspective, the substrate selectively binds *Address correspondence to this author at the Dept. of Biochemistry and Molecular Biology, Medical Institute, P. O. Box 2619, Beijing 100068, PR China; Tel: 0086-10-68789113; Fax: ?????????????????????; E-mail: [email protected] 0929-8665/11 $58.00+.00

to one of many conformations of an enzyme (or conformational ensemble), co-existing in solution, then enzymesubstrate complex undergoes serial conformational change by conformation selection and adjustment mechanism, finally the high affinity state between enzyme and substrate can be formed. The concept of “promoting motion” in enzyme activity was proposed by Benkovic and HammesSchiffer (14). They argued that enzyme activity results from the coupled motions of some residues (promoting motions) of an enzyme, but they did not identify the essential relationship between promoting motion and the conformational changes in an enzyme. There is substantial evidence for this concept, but debate about it continues (15, 16). In my view, all these fragmentary ideas about enzyme mechanisms are constructed on the basis of experimental observations and experimental models that are not logically compatible with each other. For example, the activation energy of enzymatic reaction is greatly decreased and protein flexibility (or internal motion of enzyme) influences enzyme activity, but none can propose a reasonable explanation why protein flexibility decrease activation energy of enzymatic reaction (5-7, 14-16). In my opinion, we need a fundamental theory of protein science that can explain the essential relationship between protein dynamics and protein conformation, and then a unified model for enzyme action could be established on that basis. Fortunately, the required theory protein thermodynamic structure theory (17) - has been proposed. It explores the essential relationship between the timescale of protein conformational change and motions of the main protein chain. Simply speaking, if protein conformational change involves more residues, its timescale will be long (17, 18). Therefore, the timescale of a protein conformational change characterizes the range or size of that change and can be utilized to analyze the relationship between different types of protein conformational change and the motions of the main chain of a protein (17, 18). A unified model of enzyme action should address the following questions of enzymology. How do coupled mo© 2011 Bentham Science Publishers Ltd.

2 Protein & Peptide Letters, 2011, Vol. 18, No. 1

tions of a protein work? Why are these motions coupled? What is the relationship between them and the conformational change of an enzyme? What is the root cause of the reduction of activation energy in an enzyme-catalyzed reaction compared with a simple chemical reaction? What is the logical relationship between the kinetic parameters of an enzymatic reaction and the conformational change of enzyme and how should it be analyzed? And so on. Now we show that these questions can be resolved, at least theoretically, by applying the principles of protein thermodynamics structure theory, and a unified model of enzyme action can be naturally established on that basis. BASIC PRINCIPLES OF ENZYMATIC REACTION According to protein thermodynamics structure theory (17, 18), a protein is not a single global thermodynamic system, but composed of many thermodynamic sub-systems, called potherses. For any property of a protein, a potherse can be theoretically isolated from the complex thermal system of this protein, and the targeted property is attributed to this potherse. Also, there are numerous types of protein conformational change and each of them represents one type of concerted motion within a protein. Protein conformational change can be characterized in terms of many physical properties such as free Gibbs energy, entropy, and timescale. Among these, the timescale is of extreme importance in our analysis of enzymatic reaction because we can estimate the number of pothers involved in the conformational change of an enzyme. One basic hypothesis of our approach is that enzymereactant complex (ERC) represents one protein molecule and the nature of its conformational changes can be described by protein thermodynamic structure theory. Once a reactant binds to the enzyme, all changes in the reactant are induced by conformational changes of the ERC. On the basis of the aforementioned principles and basic facts of enzymology, we formulate the dynamic model of enzyme action as follows: 1) The enzymatic reaction results from serial conformational changes in the ERC. In other words, enzymatic reactions involve multiple kinetic steps (19). 2) Any step in an enzymatic reaction and any combination of these steps (even the whole reaction) corresponds to one type of protein conformational change (or potherse) of the ERC, which represents one type of concerted motion (or promoting motion of enzyme activity). 3) The nature of the conformational change in any step of an enzymatic reaction can be analyzed in terms of the thermodynamic parameters of the reaction, such as Km (Michaelis constant), Kd (dissociation constant between enzyme and substrate), and Kcat (turnover number) (1). 4) A protein shows numerous types conformational change that may have positive or negative effects on enzyme activity (17, 20). 5) The protein conformational change involved in an enzymatic reaction is global, not limited to the active area of the enzyme. “Global” in this context does not imply

Qinyi Zhao

6) that all parts of the enzyme are involved in the reaction, but indicates that promoting motions are sporadically distributed among different parts of the enzyme. 7) Ranging from zero to 130 kcal/mol (the maximum activation energy of protein denaturation) (21), the energy of a protein conformational change, which comes from thermal fluctuations or the hydrolysis of ATP, is the source for the enzymatic reaction. 8) A change of protein flexibility, e.g. on binding to a ligand, can modulate the dynamic properties of the protein, and then can influence enzyme activity (5, 19). The generic relationships among these different aspects of an enzymatic reaction are represented in Fig. (1) according to the protein thermodynamic structure theory. TIMESCALE OF PROTEIN CONFORMATIONAL CHANGE The size or scope of a protein conformational change can be analyzed over time and the duration of the timescale lengthens if many residues (or pother) are involved (17, 18). The timescale of protein conformational change has been extensively studied and summarized by many scientists (2224). A global protein conformational change is induced by rotation of dihedral angle (or pothers) along the main chains. In many cases, side chain interaction and movement can induce the change of global conformations because these interactions can couple with protein dynamics of main chain of a protein. So, we can theoretically analyze protein global conformational change by protein dynamics of main chain. Normally, enzyme activity is related to two different conformational changes in the enzyme. The first refers to the global conformational change related to enzyme substrate binding and formation of the transition state. The second refers to subtle conformational changes related to the breakage of chemical bonds (7). The global conformational change in the ERC results in a protein conformation that favors the formation of the transition state of the ERC, and subtle conformational changes result in the breakage of chemical bonds. The rate of subtle conformational change in an enzymatic reaction is predominantly determined by its activation energy rather than the dynamic nature of the enzyme, and it will not be discussed in detail in this paper. On the basis of this recognition, the timescales of enzymatic events and their relation to enzymatic activities are summarized in Fig. (2). Most, if not all, texts concerning enzymology interpret enzymatic mechanisms using transition state theory and only discuss the active site of the enzyme. This can be misleading and result in the belief that enzyme activity is only related to the conformation (flexibility) or the fast motions of the active site of the enzyme (5-7). Given that the timescale and spatial scale of fast motion of side chains of a protein are small and that the timescale of enzymatic reactions is large, we conclude that the ratelimiting motion of enzyme activity cannot be due to the fast motion of residues of the active site of enzyme. This conclusion is supported by the following facts:

Dynamic Model for Enzyme Action

Protein & Peptide Letters, 2011, Vol. 18, No. 1

3

Fig. (1). The logical relationships among different concepts of enzymatic reactions. The timescale of protein conformational change is of great importance within protein thermodynamic structure theory for it represents the size or range of protein conformational change. The explanation of allodynamic regulation can be found in following discussion.

Fig. (2). Time scale of protein conformational changes involved in enzymatic reactions and their relationship. Theoretically, protein conformational changes can be classified into two categories: subtle conformational change (induced by fast motion of side chains) and global conformational change (induced by motion of the backbone of the main chain). According to protein thermodynamic structure theory (17, 18), the maximum rate of global protein conformational change is approximately 109, and it represents the upper limit of Kcat of enzymes.

1) Global conformational change is required for enzyme activity (25). 2) Enzyme activity can be influenced by the viscosity of the solvent (26). A physical study demonstrates that local protein dynamics are independent of the solvent and that global protein conformational change is sensitive to it (27). 3) The binding of oxygen to hemoglobin can induce global conformational change on the entire structure of the latter (28). Moreover, the regulatory site, which is distant from the active site of the enzyme, can influence enzyme activity. DYNAMIC SITES OF ENZYME ACTIVITY There is a relationship between the timescale of protein conformational change and the coupled motions of the main chains, and the number of pothers for one type of conformational change can be estimated on the basis of its timescale (17, 18). In a simple model in which the energy difference between different protein conformations can be neglected, the number of pothers involved in enzyme activity can be calculated by the following equations (17, 18): T=S/v S=N!

where T represents the timescale of the enzymatic process (or enzymatic potherse), S represents the protein conformational space of the enzymatic potherse, v is approximately 1010 turn/second (rate of conformational change), and N is the number of pothers involved in the enzyme activity. The turnover of an enzyme-catalyzed reaction is approximately 103-107 per second (1). The timescales (T) for enzymatic reactions are approximately 10-7 - 10-3 second. Therefore we estimate that 6-10 pothers of a protein are directly involved in enzyme activity. The sites which involve protein conformational change of enzymatic reaction is called dynamic site of enzymatic reaction, which corresponds to promoting motion of enzymatic reaction. Fig. (3) explains general profile of dynamic sites of protein conformational change. This theory cannot predict the detailed positions of these promoting motions in a protein, but can predict that the dynamic sites of enzyme activity will be distributed in the main chain and cannot be concentrated in one site only. Enzymes with a low Kcat value are involved in large conformational changes and vice versa. Therefore, it is predicted that urease, which has a high Kcat value, is involved in small conformational changes (1). The work of Henzler-Wildman et al. supports our prediction. The authors found that six amino acid residue substitutions of mesoAdk with the residue of thermoAdk mimicked


the protein flexibility of thermoAdk (29). Further evidence comes from a study concerning RNase A in which the authors found that the internal motions related to RNase A activity are predominantly located in five sites (residues 12, 43, 86, 102, 121) (30).

Qinyi Zhao

For the Ser214 substitution in thrombin, an evolutionary marker for the serine protease family (33), the K1 of the mutant enzymes ranges from 100 to 0.036 M-1 s-1 and K2 from 9 to 46 s-1. The residue substitution has different effects on K1 and K2, and can have opposing effects on these parameters (S214D vs. Wt). Therefore, protein flexibility is related to substrate binding (K1) and the catalytic ability of the enzyme (K2) is governed by different sites. THERMODYNAMIC NATURE OF ENZYMATIC REACTIONS According to the kinetic parameters of the serine protease reaction, the time characteristics of the reaction are presented in Fig. (4).

Fig. (3). Dynamic sites of protein conformational change. A type of protein conformational change of enzymatic reaction results from the coupled motions (or rotations) of many dynamic sites of a protein. The dynamics site may be located out off active site (or binding site) of an enzyme.

An S-S bond introduced into T4 lysozyme greatly decreases protein flexibility and enhances the stability of the protein under high temperature conditions; it has little to do with the activity profile of the enzyme (31). This suggests that changes induced by S-S bonds do not relate to the dynamic sites of lysozyme activity. The unstructured area (loop, hinge) of a protein has the highest flexibility and dynamic sites of enzyme activity can be located in such areas. Some experimental data support this conclusion. A mutation in Loop 52-72, which is distant from the active site of the enzyme, influences the activity of fructose-1,6-bisphosphatase (32). KINETICS PARAMETERS OF ENZYMATIC REACTIONS AND PROTEIN CONFORMATIONAL CHANGES Conformational change can be analyzed by the kinetic parameters of enzymatic reactions. Normally, enzymecatalyzed reactions e.g. of serine protease are represented as follows (33):

Km, K1, K2, K-1, K3 are kinetic parameters for the enzyme-catalyzed reaction. In this case, the E represents enzyme, EP represents intermediate complex between enzyme and one production, and P represents production (33). If the conformational change involved in the enzymecatalyzed reaction occurs in a large space and protein flexibility is low, the kinetic parameters will have low values. This rule can be utilized to analyze the change in kinetic parameters among substrate analogs or for residue substitutions. It is well established that binding between proteins is very slow but occurs with high affinity.

The rate-limiting step for serine protease is the formation of the ES complex, rather than breakage of chemical bonds when K2 is greater than K-1, where they represent rate constants of protein conformational changes in opposite directions. Consequently, the conversion of chemical bonds may not be at the peak of the energy curve of the enzymatic reaction. The traditional transition state theory can satisfactorily describe the nature of elementary reactions, but not the multiple steps of an enzymatic reaction. The time scale of a single enzymatic reaction (10-7 -10-2 second) is longer than that of a chemical reaction (usually less than 10-12 second). Enzymatic reactions are not closed thermodynamic systems, unlike simple chemical reactions, and there is heat exchange between the ES complex and the environment. This is a fundamental difference between enzymatic and elementary reactions in chemistry. Protein conformational changes can occur in any direction and do not always coincide with the enzymatic reaction. The total energy decreases, but the energy along the transition state may be increased. This suggests that the conformation of the ES complex, as well as the organization of the bonds within the complex, is similar to the transition state of the chemical reaction, i.e. the energetic pre-organization concept (8, 9) is upheld for enzyme-substrate binding. In this way, some of activation energy of a chemical reaction can be transformed (or dispersed) into binding energy of multiple weak bonds between the substrate and enzyme. The similarity between the ES complex and the transition state provides a thermodynamic explanation for the high efficiency of this enzyme, which has long been puzzling (2, 7, 8). It can also be analyzed mathematically. According to the transition state theory for an elementary reaction, Ke-G/RT=e-G1/RTe-G2RTe-G3/RTe-G4/RTe-G5/RTe-G6/RT G= G1+G2+G3+G4+G5+G6 Where K represents the rate constant of the reaction, G represents the activation energy of the reaction, and it is assumed that G can be divided into six parts (or processes). If all these parts are independent of one another, as is the case in simple chemical reactions, the equations will be validated for all cases. However, in enzymatic reactions the independence of these parts has been lost as they do not occur synchronously and there is heat exchange between the ES complex and the


Protein & Peptide Letters, 2011, Vol. 18, No. 1

5

Fig. (4). Two models of enzymatic reactions E represents energy, T represents time and S represents the similarity between the ES complex and the transition state of ES. (A) Traditional transition state model of enzymatic mechanisms. ES represents the enzyme-substrate complex. E represents energy and T represents time. This model originates from transition state theory developed in the study of elementary chemical reactions. (B) Time characteristics of enzyme-catalyzed reactions (or conformational change of ES complex) for serine protease according to our model. In this case, K2 is greater than K -1. After binding between the enzyme and substrate, the ES complex undergoes a conformational change and transforms into the EP complex. ES, EP and TS (transition state) represent several temporal states of the ES complex. Some types of conformational change have high activation energies and they are rate-limiting steps in an enzymatic reaction. In some cases, the activation energy of a conformational change of ES (E-1, related to K-1) in the reverse direction is higher than that of a conformational change in the positive direction (E2, related to K2) or formation of the transition state. (C) Time characteristics of enzyme-catalyzed reactions along transition state coordinates are presented in this figure. Although the Gibbs free energy decreases in the enzyme-catalyzed process, the energy level of the ES complex increases along the transition state coordinates. The ES complex can present with different conformations and enzyme-catalyzed reactions can occur in different ways (dashed and real line). (ES)* represents the enzyme conformation in the transition state.

environment. Some dynamic processes of a chemical reaction can be transformed into kinetic processes and do not contribute to the activation energy of the reaction. The rate constant of an enzymatic reaction can be written as: Ke a1a2a3a4 e-G5/RT e-G6/RT The Arrhenius equation for an enzymatic reaction can be written as: V=(A’ a1a2a3a4) e-(G5+G6)/RT where A’ represents the geometrical factor for a simple chemical reaction, and an represents the contribution of part n to the pre-exponential factor of the Arrhenius equation. Therefore, the activation energy of the enzymatic reaction (G5+G6) is much lower than that of the chemical reaction (G). As GB (binding energy between enzyme and substrate) represents the maximal value of enzyme-substrate interactions, the following equation could be obtained: GB=-RTlnKB

is approximately 10-4). Dramatic changes in the A factor (pre-exponential factor of Arrhenius equation) have been discussed by JP Klinman (19). This view provides a reasonable explanation for the role of protein dynamics in enzyme activity (14, 15, 16). The promoting motions (14) of enzyme activity have no impact on the reduction of activation energy of an enzymatic reaction, but represent dynamic processes of conformational change involved in the formation of the transition state of the ERC, and this is in agreement with A Warshel (16). In other way, the modulating protein flexibility can influence fine structure of transition state of enzymatic reaction and thus lower the activation energy of enzymatic reaction (34, 35). TIGHT ENZYME BINDING TO TRANSITION STATE SUBSTRATES IS UNNECESSARY According to transition state theory and conventional view of enzymology (1), enzymatic reactions can be represented with the following formulation:

Km1/KB G1+G2+G3+G4GB Where K B represents binding constant, Km represents Michaelis constant of enzymatic reaction. From these equations, we can find that the conformation of the ES complex is similar to the transition state of the ERC in some degree. The high efficacy of enzyme activity comes from its affinity to substrate and binding energy between enzyme and substrate is a part of activation energy of chemical reaction. Thus, KB represents maximum value an enzyme to accelerate a chemical reaction. For a serine protease (33), the high affinity between enzyme and substrate can theoretically accelerate their target reactions by 104-fold (Km

By studying the relationships of kinetic parameters in enzymatic reactions expressed in this formulation, one can demonstrate that an enzyme binds tightly to the transition state substrate (S*). However, this is not the case. By applying the principles of the dynamic model of enzyme activity, we formulate the relationship among different steps of the enzymatic reaction as follows:


The transition conformation of enzymes (E*) differs from that of naked enzymes and enzyme-substrate complex (ES)’. Therefore, we conclude it is unnecessary to suppose that enzymes bind tightly to their transition state substrates (2). According to protein thermodynamic structure theory, (ES)’ and (ES)* represent two different potherses, the labeling parameters (here refers Km or Kcat) for them differs from each other and there is no relationship between them. The experimental evidence is that Km and Kcat of an enzymatic reaction varies from substrate to substrate and logical relationship between Km and Kcat of enzymatic reactions has never been reported. ALLODYNAMIC REGULATION MECHANISM VS. ALLOSTERIC REGULATION OF ENZYME ACTIVITY The dynamic model of enzyme activity reveals another mechanism of regulation, which we have proposed previously (17) and called the ‘allodynamic regulation mechanism’. It proposes that enzyme (or receptor) activity can be regulated by modifying the internal motions (or dynamic properties) at dynamic sites of enzyme activity. This view, although different from the conventional view of allosteric regulation, was supported from studies concerning single ion channels. For example, pentobarbital, an antagonist of the GABA receptor, produces its effect by modifying the frequency of the opening of the GBBA receptor but has no effect on the constant unitary conductance of the GABA receptor (36, and references therein). This indicates that receptorantagonist binding does not induce structural changes in the gate area of a channel, and that can be explained by the allodynamic mechanism of regulation. Compared with the allosteric regulation mechanism, allodynamic regulation has the advantage that it provides a simple and reasonable explanation for the efficiency and complex interactions of different regulators and agonists on receptor activity, which are of great importance in signal regulation (16, 37). Dynamic changes in a protein can induce structural change but not always. Therefore, a structural change in a protein is unnecessary in the allodynamic regulation mechanism, but it will occur in most cases to some degree, concomitantly with a change in the protein dynamics. Therefore, the allosteric regulation mechanism reflects structural features of the allodynamic regulation mechanism. However, for the cooperation phenomenon observed in hemoglobin-O2 binding, the structural change in hemoglobin is a necessity (28). From the viewpoint of experimental science, it is difficulty to distinguish dynamic changes from structural changes in a protein. Critical evidence for the allodynamic regulation mechanism has not been reported in enzymology, but it can explain experimental results. The logical separation of different

Qinyi Zhao

properties of a protein, as we have emphasized previously (17), can be utilized to distinguish allodynamic from allosteric regulation mechanisms. For example, separate insertions of proline at positions 50 and 51 in loop 52-57 of porcine fructose-1,6-bisphosphatase reduces kcat up to 3-fold, with no effect on the Km for fructose 1,6-bisphosphate (32). In this case, the change in Km is separate from the change in kcat. If allosteric regulation governed the behavior then the changes in K m and kcat would coincide. The M24 and G121 of dihydrofolate reductase are two dynamic sites of enzymatic reaction (38).The mutation of these residues greatly modulate protein mobility (or protein flexibility) at these sites and impair enzyme activity, but have no impact on protein three-dimensional structure (38). Numerous facts related to allodynamic regulation have been discussed by many scientists (39-44, and references therein). Our task was to provide a theoretical model for these. It seems clear from the exploration in this paper that hypotheses arising from the protein thermodynamic structure theory explain these aspects of allodynamic regulation and also explain the nature of enzyme catalysis and its relationship to dynamic conformational changes in proteins. PROTEINS FLEXIBILITY AND ENZYMES ACTIVITY In my opinion, the alteration of enzyme activity induced by urea (5) and regulation of receptor activity by volatile anesthetics (37, 45) are carried out by allodynamic mechanism. When protein flexibility is enhanced by urea, protein dynamics at many local sites of a protein is altered (46-49). This will result in the change of enzyme activity. The generic relationship between enzyme activity and protein flexibility is represented in Fig. (5). Our conclusion is supported by following facts: 1) Logical separation of binding ability of enzyme and substrate. It has shown that the binding ability between enzyme and substrate is independent to enzyme activity (50). 2) Logical separation of protein structure and enzyme activity (5, 34, 35). Also see Fig. (5). 3) Cooperation between temperature and urea on enzyme activity (34, 35). This indicated that the change of enzyme activity is induced by protein flexibility, rather than protein conformation. 4) The rate of enzyme inactivation is greater than that of protein denaturation (51). 5) At low concentration of protein denaturants, the change of protein dynamics occurs at residues of hydrogen exchange core, which are not in active site of enzymatic reaction (52, 53). It also provides powerful evidence that alteration of enzyme activity in the presence of urea is induced by global conformational change, not local conformational change at active site. Some may argue that the alteration of enzyme activity induced by urea results from subtle conformational change of enzyme at active site (5). If a conformational change occurred, a sudden change of parameters of enzymatic reaction,


such as activation energy, should be observed (17). However, it is not a common phenomenon and cannot account for all natures of enzyme activity. The change of residue motion has been observed for a number of enzymes (e.g. 41).

Protein & Peptide Letters, 2011, Vol. 18, No. 1 [14] [15] [16] [17] [18] [19] [20] [21] [22]

Fig. (5). Generic relationship between protein flexibility and enzyme activity. Within this figure, A represents enzyme activity and GS represents global structure of enzyme. The logical separation between enzyme activity and protein structure can be clearly seen.

CONFLICT OF INTEREST The author declares that he has no conflict of interest. ACKNOWLEDGEMENT I would like to thank Professor VV Matveev (Laboratory of Cell Physiology, Institute of Cytology, Russian Academy of Sciences, Petersburg, Russia) for his thoughtful and critical comments and suggestions in revising the manuscript. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

Fersht, A. Enzyme Structure and Mechanism. Freeman press: San Francisco, 1985. Pauling, L. Molecular architecture and biological reactions. Chem. Eng. News., 1946, 24, 1375-1377. Koshland, D.E. Application of a Theory of Enzyme Specificity to Protein Synthesis. Proc. Natl. Acad. Sci. U. S. A., 1958, 44, 98-104. Koshland, D.E. The key-lock theory and the induced fit theory. Angewandte Chemie-international edition., 1995, 33, 2375-2378. Tsou, C.L. Active Site Flexibility in Enzyme Catalysis. Ann. N. Y. Acad Sci., 1998, 864, 1-8. Yuan, Z.; Zhao, J.; Wang, Z.X. Flexibility analysis of enzyme active sites by crystallographic temperature factors. Protein Eng., 16, 109-114 (2003). Schwartz, S.D.; Schramm, V.L. Enzymatic transition states and dynamic motion in barrier crossing. Nat. Chem. Boil., 2009, 5, 551558. Warshel, A.; Levitt, M. Theoretical studies of enzymatic reactions: dielectric electrostatic and steric stabilization of the carbonium Ion in the Reaction of Lysozyme. J. Mol. Biol., 1976, 103, 227-249. Cannon, W.R.; Benkovic, S.J. Solvation, Reorganization Energy, and Biological Catalysis. J. Biol. Chem., 1998, 273, 26257-26260. Frauenfelder, H. Proteins: Paradigms of complexity. Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 2479-2480. Teague, S.J. Implications of protein flexibility for drug discovery. Nat. Rev. Drug Discov., 2003, 2, 527-41. Csermely, P.; Palotai, R.; Nussinov, R. Induced fit, conformational selection and independent dynamic segments: an extended view of binding events. Trends Biochem Sci. 2010, doi:10.1016/j.tibs. Grünberg, R.; Leckner. J.; Nilges. M. Complementarity of structure ensembles in protein–protein binding. Structure, 2004, 12, 2125– 2136

[23] [24] [25] [26] [27] [28] [29] [30] [31] [32]

[33] [34]

[35]

[36] [37] [38]

[39] [40]

7

Benkovic, S.J.; Hammes-Schiffer, S. A Perspective on Enzyme Catalysis. Science, 2003, 301, 1196-1202. Garcia-Viloca, M.; Gao, J.; Karplus, M.; Truhlar, D.G. How Enzymes Work: Analysis by Modern Rate Theory and Computer Simulations. Science, 2004, 303, 186-195. Kamerlin, S.C.; Warshel, A. At the dawn of the 21st century: Is dynamics the missing link for understanding enzyme catalysis? Proteins, 2010, 78, 1339-75. Zhao, Q. Protein thermodynamic structure. IUBMB life, 2009, 61, 600-606. Zhao, Q. Irreversible thermodynamics theory for protein folding and protein thermodynamics structure. Prog Biophys Biochem., 2001, 28, 429-435. Klinman, J.P. Linking protein structure and dynamics to catalysis: the role of hydrogen tunneling. Phil. Trans. R. Soc. B., 2006, 361, 1323-1331. Hammes, G.G. Multiple conformational changes in enzyme catalysis. Biochemistry, 2002, 41, 8221-8. Kreimer, D.I.; Shnyrov, V.L.; Villar, E.; Silman, I.; Weiner, L. Irreversible thermal denaturation of Torpedo californica acetylcholinesterase. Protein Sci., 1995, 4, 2349–2357. McCammon, J.A. Protein Dynamics. Rep. Prog. Phys., 1984, 47, 146 Teilum, K.; Olsen, J.G.; Kragelund, B.B. Functional aspects of protein flexibility. Cell Mol. Life Sci., 2009, 66, 2231-47. Parak, F.G.; Achterholda, K.; Schmidta, M.; Prusakov, V.; Crocia, S. Protein dynamics on different timescales. J. Non-Cryst. Solids, 2006, 352, 4371-4378. Gallagher, S.C.; Callaghan, A.J.; Zhao, J.; Dalton, H.; Trewhella, J. Global conformational changes control the reactivity of methane monooxygenase. Biochemistry, 1999, 38, 6752-60. Barbier, G.G.; Campbell, W.H. Viscosity Effects on Eukaryotic Nitrate Reductase Activity. J. Biol. Chem., 2005, 280, 2604926054. Fenimore, P.W.; Frauenfelder, H.; McMahon, B.H.; Parak. F.G. Slaving solvent fluctuations dominate protein dynamics and functions. Pro. Natl. Acad. Sci. U. S. A., 2002, 99, 16047-16051. Perutz, M.F. Mechanisms of cooperativity and allosteric regulation in proteins. Cambridge University Press: London, 1990. Henzler-Wildman, K.A.; Lei, M.; Thai, V.; Kerns, S.J.; Karplus, M.; Kern, D. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature, 2007, 450, 913-916. Watt, E.D.; Shimada, H.; Kovrigin, E.L.; Loria, J.P. The mechanism of rate-limiting motions in enzyme function. Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 11981-11986. Wetzel, R.; Perry, L.J.; Baase, W.A.; Becktel, W.J. Disulfide bonds and thermal stability in T4 lysozyme. Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 401-405. Nelson, S.W.; Choe, J.Y.; Honzatko, R.B.; Fromm, H.J. Mutations in the Hinge of a Dynamic Loop Broadly Influence Functional Properties of Fructose-1,6-bisphosphatase. J. Biol. Chem., 2000, 275, 29986-29992. Krem, M.M.; Prasad, S.; Cera, E.D. Ser214 is crucial for substrate binding to serine proteases. J. Biol. Chem., 2002, 277, 40260– 40264. Zoldák, G.; Sut'ák R.; Antalík M.; Sprinzl, M.; Sedlák, E. Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus. Eur. J. Biochem., 2003, 270, 48874897 Zoldák, G.; Sprinzl, M.; Sedlák, E. Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions. Eur. J. Biochem., 2004, 271, 48-57. Hammond, C. Cellular and molecular neurophysiology, Academy press: London, 2008. Wang, A.; Zhang, Z.; Zhao, Q. In vivo measurement of protein functional changes. Int. J. Biol. Sci., 2009, 5, 411-420. Agarwal, P.K.; Billeter, S.R.; Rajagopalan, P.T.R.; Benkovic, S.J.; Hammes-Schiffer, S. Network of coupled promoting motions in enzyme catalysis. Pro. Natl. Acad. Sci. U. S. A., 2002, 99, 2794– 2799. Wand, J.A. On the dynamic origin of allosteric activation. Science, 2001, 24, 1395-1315. Popovych, N.; Sun, S.; Ebright, R.H.; Kalodimos, C.G. Dynamically driven protein allostery. Nat. Struct. Mol. Biol., 2006, 13, 831-8.

8 Protein & Peptide Letters, 2011, Vol. 18, No. 1 [41] [42] [43] [44] [45] [46]

[47]

Qinyi Zhao

Gunasekaran, K.; Ma, B.; Nussinov, R. Is allostery an intrinsic property of all dynamic proteins? Proteins, 2004, 57, 433–443. Kern, D.; Zuiderweg, E.R. The role of dynamics in allosteric regulation. Curr. Opin. Struct. Biol., 2003, 13, 748-57. Cui, Q.; Karplus, M.; Allostery and cooperativity revisited. Protein Sci., 2008, 17, 1295-307. Goodey, N.M.; Benkovic, S.J. Allosteric regulation and catalysis emerge via a common route. Nat. Chem. Biol., 2008, 4, 474-82. Zhao, Q. Protein flexibility as biosignal. Crit. Rev. Eukaryot. Gene. Expr. In press Krishna, M.M.G.; Maity, H.; Rumbley, J.N.; Lin, Y.; Englander, S.W. Order of steps in the cytochrome c folding pathway: Evidence for a sequential stabilization mechanism. J. Mol. Biol., 2006, 359, 1410-1419. Maity, H.; Lim, W.K.; Rumbley, J.N.; Englander, S.W. Protein hydrogen exchange mechanism: Local fluctuations. Protein Sci., 2004, 12, 153-160.

,Received: ????????????

Revised: ????????????

Accepted: ????????????

[48]

[49] [50] [51] [52] [53]

Vogtt, K.; Winter, R. Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance. Braz. J. Med. Biol. Res., 2005, 38, 1185-93. Bruix, M.; Ribó, M.; Benito, A.; Laurents, D.V.; Rico, M.; Vilanova, M. Destabilizing mutations alter the hydrogen exchange mechanism in ribonuclease A. Biophys. J., 2008, 94, 2297-305. Nelson, C.A.; Hummel, J.P. Reversible denaturation of pancreatic ribonuclease by urea. J. Biol. Chem., 1962, 237, 1567-74. Yao, Q.Z.; Tian, M.; Tsou, C.L. Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation. Biochemistry, 1984, 23, 2740-2744. Li, R.; Woodward, C. The hydrogen exchange core and protein folding. Protein Sci., 1999, 8, 1571-90. Spudich, G.; Lorenz, S.; Marqusee, S. Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state. Protein Sci., 2002, 11, 522-8.