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• W2027747381 abstract "A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia colidihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1–4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate inE. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of α carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following α helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates. A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia colidihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1–4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate inE. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of α carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following α helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates. circularly permuted dihydrofolate DHF reductase tetrahydrofolate trimethoprim The active site of an enzyme contains amino acid residues involved in enzyme function. Some residues in the active site bind to the substrate or cofactor, and others are involved in the catalysis itself. In addition, residues away from the active site sometimes promote the catalytic reaction through intramolecular interactions (1Miller G.P. Benkovic S.J. Biochemistry. 1998; 37: 6327-6335Crossref PubMed Scopus (64) Google Scholar, 2Miller G.P. Benkovic S.J. Biochemistry. 1998; 37: 6336-6342Crossref PubMed Scopus (59) Google Scholar). Enzyme-catalyzed reactions proceed, in general, through multiple steps, including binding of the substrate, catalysis, and release of product. At each step, certain amino acid residues play critical roles. However, an isolated collection of these directly functioning residues is not sufficient to obtain catalytic activity. Amino acid residues should be connected covalently to make up a polypeptide chain with a specific amino acid sequence that determines a proper tertiary structure. The functioning residues on the peptide backbone are then arranged to give the proper configuration for effective catalytic activity. Because enzymes cleaved at certain sites have been demonstrated to show catalytic activity as high as that of uncleaved enzyme (3Luger K. Hommel U. Herold M. Hofsteenge J. Kirschner K. Science. 1989; 243: 206-210Crossref PubMed Scopus (191) Google Scholar, 4Buchwalder A. Szadkowski H. Kirschner K. Biochemistry. 1992; 31: 1621-1630Crossref PubMed Scopus (74) Google Scholar, 5Protasova N.Y. Kireeva M.L. Murzina N.V. Murzin A.G. Uverski V.N. Gryaznova O.I. Gudkov A.T. Protein Eng. 1994; 7: 1373-1377Crossref PubMed Scopus (43) Google Scholar, 6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar, 7Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11980-11984Crossref PubMed Scopus (47) Google Scholar, 8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar, 9Horlick R.A. George H.J. Cooke G.M. Tritch R.J. Newton R.C. Dwivedi A. Lischwe M. Salemme F.R. Weber P.C. Horuk R. Protein Eng. 1992; 5: 427-431Crossref PubMed Scopus (32) Google Scholar, 10Zhang T. Bertelsen E. Benvegnu D. Alber T. Biochemistry. 1993; 32: 12311-12318Crossref PubMed Scopus (82) Google Scholar, 11Hahn M. Piotukh K. Borriss P. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10417Crossref PubMed Scopus (77) Google Scholar, 12Mullins L.S. Wesseling K. Kuo J.M. Garrett J.B. Raushel F.M. J. Amer. Chem. Soc. 1994; 116: 5529-5533Crossref Scopus (19) Google Scholar, 13Viguera A.R. Blanco F.J. Serrano L. J. Mol. Biol. 1995; 247: 670-681Crossref PubMed Scopus (126) Google Scholar, 14Kreitman R.J. Puri R.K. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6889-6893Crossref PubMed Scopus (105) Google Scholar, 15Vignais M.L. Corbier C. Mulliert G. Branlant C. Branlant G. Protein Sci. 1995; 4: 994-1000Crossref PubMed Scopus (18) Google Scholar, 16Kommar A.A. Jaenicke R. FEBS Lett. 1995; 376: 195-198Crossref PubMed Scopus (39) Google Scholar), chain connectivity must not be absolutely required for catalytic activity. This means that chain connectivity is crucial for catalytic activity in some regions, but in others, it is not. The detailed configuration of the functioning residues seems to depend on local peptide backbone rather than the overall polypeptide chain. In this paper, we define a “functional element” as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. Such functional elements would be distributed across the primary structure. When the enzyme is properly folded, these elements would associate to make a functional active site (Fig. 1). Identifying such functional elements would be very informative in revealing the architecture of enzyme function. Here, we propose a novel approach for identifying the functional elements of an enzyme.Based on our definition of a functional element, alteration of main chain connectivity (peptide bond cleavage) seems to be a useful approach for locating functional elements. Possible consequences of peptide bond cleavage are illustrated in Fig. 1. If the main chain is cleaved within a region that is indispensable for protein folding (designated as the folding element), the resulting protein will not be able to fold to a stable conformation (case 1). In the other cases, the properties of the cleaved protein would vary depending on the location of the cleavage site. Cleavage within a functional element (case 2) will break the local configuration, leading to loss of the function contributed by the element, whereas those outside (case 3) or at the boundary of (case 4) the functional elements will not, resulting in a minor effect on the enzyme function. Even if functional elements are located side by side, distribution of the functional elements can be recognized by the presence of the boundaries (compare case 2 and case 4). Based on this idea, systematic peptide bond cleavage and characterization of the products are crucial to locating functional elements. The simplest way to cleave a peptide bond is to break the polypeptide chain into two fragments by manipulation of the coding gene. However, this dissection method is poorly suited for the purpose of obtaining a variety of cleaved proteins without disrupting the overall tertiary fold, because production of properly folded protein requires the association of the fragmentary chains by long-range interaction. Circular permutation analysis, in which the original N and C termini of a protein are connected by an appropriate linker and new termini are created at a position of interest (Fig.1), can overcome such entropic problems because it allows a peptide bond to be broken without fragmenting the protein into two pieces. To date, a number of circularly permuted proteins with various folded structures have been reported (3Luger K. Hommel U. Herold M. Hofsteenge J. Kirschner K. Science. 1989; 243: 206-210Crossref PubMed Scopus (191) Google Scholar, 4Buchwalder A. Szadkowski H. Kirschner K. Biochemistry. 1992; 31: 1621-1630Crossref PubMed Scopus (74) Google Scholar, 5Protasova N.Y. Kireeva M.L. Murzina N.V. Murzin A.G. Uverski V.N. Gryaznova O.I. Gudkov A.T. Protein Eng. 1994; 7: 1373-1377Crossref PubMed Scopus (43) Google Scholar, 6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar, 7Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11980-11984Crossref PubMed Scopus (47) Google Scholar, 8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar, 9Horlick R.A. George H.J. Cooke G.M. Tritch R.J. Newton R.C. Dwivedi A. Lischwe M. Salemme F.R. Weber P.C. Horuk R. Protein Eng. 1992; 5: 427-431Crossref PubMed Scopus (32) Google Scholar, 10Zhang T. Bertelsen E. Benvegnu D. Alber T. Biochemistry. 1993; 32: 12311-12318Crossref PubMed Scopus (82) Google Scholar, 11Hahn M. Piotukh K. Borriss P. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10417Crossref PubMed Scopus (77) Google Scholar, 12Mullins L.S. Wesseling K. Kuo J.M. Garrett J.B. Raushel F.M. J. Amer. Chem. Soc. 1994; 116: 5529-5533Crossref Scopus (19) Google Scholar, 13Viguera A.R. Blanco F.J. Serrano L. J. Mol. Biol. 1995; 247: 670-681Crossref PubMed Scopus (126) Google Scholar, 14Kreitman R.J. Puri R.K. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6889-6893Crossref PubMed Scopus (105) Google Scholar, 15Vignais M.L. Corbier C. Mulliert G. Branlant C. Branlant G. Protein Sci. 1995; 4: 994-1000Crossref PubMed Scopus (18) Google Scholar, 16Kommar A.A. Jaenicke R. FEBS Lett. 1995; 376: 195-198Crossref PubMed Scopus (39) Google Scholar). Construction of circularly permuted (CP)1 variants has revealed that the N and C termini of proteins can be moved to alternative positions without lethal damage and that the order of peptide synthesis is not critical for the final tertiary structure of a protein. In most cases, newly created termini were directed to sites such as interdomain hinges or surface loops. On the other hand, random circular permutation was applied to the catalytic chains of aspartate transcarbamoylase to investigate possible rules for inserting termini in various regions of the three-dimensional structures of protein (8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar). In the same way that alanine scanning has been used to elucidate the role of side chains (17Cunningham B.C. Wells J.A. Science. 1989; 244: 1081-1085Crossref PubMed Scopus (1080) Google Scholar), systematic circular permutation analysis will be useful for estimating the role of main chain connectivity.We chose an active site flexible loop of Escherichia colidihydrofolate reductase (DHFR) (EC 1.5.1.3) as the target of an investigation of functional elements. DHFR catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) and plays an important role in supplying the cofactor for one-carbon transfer reactions, e.g. the reaction catalyzed by thymidylate synthase (18Blakley R.L. Blakley R.L. Benkovic S.J. Dihydrofolate Reductase in Folates and Pteridines. 1. Wiley, New York1984: 191-253Google Scholar). The flexible loop connecting β strand A and α helix B of E. coli DHFR (Ala-9∼Leu-24) has been called loop I (19Volz K.W. Matthews D.A. Alden R.A. Freer S.T. Hansch C. Kaufman B.T. Kraut J. J. Biol. Chem. 1982; 257: 2528-2536Abstract Full Text PDF PubMed Google Scholar) or the M20 loop (20Bystroff C. Oatley S.J. Kraut J. Biochemistry. 1990; 29: 3263-3277Crossref PubMed Scopus (266) Google Scholar) (Fig.2). This loop has attracted much attention because of its variable conformations in crystal structures (20Bystroff C. Oatley S.J. Kraut J. Biochemistry. 1990; 29: 3263-3277Crossref PubMed Scopus (266) Google Scholar, 21Bystroff C. Kraut J. Biochemistry. 1991; 30: 2227-2239Crossref PubMed Scopus (203) Google Scholar). Sawaya and Kraut (22Sawaya M.R. Kraut J. Biochemistry. 1997; 36: 586-603Crossref PubMed Scopus (604) Google Scholar) summarized the tertiary structures of the enzymes in various ligation states and provided snapshots of the enzymes at each step of the catalytic cycle. They observed that a conformational oscillation of the M20 loop occurred as part of the catalytic cycle. The catalytic role played by the M20 loop was examined by the use of a deletion mutant in which the hairpin-forming residues (Met-16∼Ala-19) had been replaced by a single glycine (23Li L. Falzone C.J. Write P.E. Benkovic S.J. Biochemistry. 1992; 31: 7826-7833Crossref PubMed Scopus (100) Google Scholar). The results suggested that those residues contributed to the acceleration of hydride transfer reaction without significantly affecting the binding of DHF and NADPH and release of THF. NMR studies indicate that the M20 loop oscillates at a frequency similar tok cat, which is limited by the release of the product, THF (24Falzone C.J. Wright P.E. Benkovic S.J. Biochemistry. 1994; 33: 439-442Crossref PubMed Scopus (146) Google Scholar, 25Fierke C.A. Johnson K.A. Benkovic S.J. Biochemistry. 1987; 26: 4085-4092Crossref PubMed Scopus (470) Google Scholar). The reaction catalyzed by E. coliDHFR proceeds through binding of DHF to the holoenzyme followed by proton and hydride transfer reaction, release of NADP+, binding of NADPH, and release of THF. Thus, the M20 loop is likely to play multiple roles. This conclusion is also supported by evidence from the deletion mutant indicating that the M20 loop seems to contribute to the hydride transfer reaction (23Li L. Falzone C.J. Write P.E. Benkovic S.J. Biochemistry. 1992; 31: 7826-7833Crossref PubMed Scopus (100) Google Scholar) and that the movement of the loop may be a limiting factor in substrate turnover (24Falzone C.J. Wright P.E. Benkovic S.J. Biochemistry. 1994; 33: 439-442Crossref PubMed Scopus (146) Google Scholar). In order to elucidate the role of the M20 loop in detail, it would be necessary to understand the flexible loop as an assembly of independent regions. Therefore, the idea of functional elements will be greatly helpful.Figure 2Drawing of the tertiary structure of DHFR using Protein Data Base file 1rx1. Four α-helices (αB, αC, αE, and αF) and eight α-structures (βA, βB, βC, βD, βE, βF, βG, and βH) are shown by ribbons. The peptide region from Leu-8 to Asp-27 is indicated by the black area. Positions of Leu-8 and Asp-27 and their side chains are shown by arrows and by balls and sticks, respectively. The positions of the N and C termini are also indicated.View Large Image Figure ViewerDownload (PPT)To this end, systematic circular permutation analysis was performed to investigate whether the M20 loop could be further divided into multiple functional elements. Circularly permuted forms of DHFR have been reported for the enzymes of mouse (4Buchwalder A. Szadkowski H. Kirschner K. Biochemistry. 1992; 31: 1621-1630Crossref PubMed Scopus (74) Google Scholar) and E. coli (5Protasova N.Y. Kireeva M.L. Murzina N.V. Murzin A.G. Uverski V.N. Gryaznova O.I. Gudkov A.T. Protein Eng. 1994; 7: 1373-1377Crossref PubMed Scopus (43) Google Scholar). In designing the peptide linker connecting the original N and C termini ofE. coli DHFR, we have shown that the five-glycine linker is the most favorable for maintaining the overall structure and function (6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar). The results obtained in this study suggested the presence of three boundaries, indicating that the M20 loop could be divided into multiple functional elements.DISCUSSIONMimicking enzyme catalysis is one of the ultimate goals of biochemistry. Among the many attempts to design an artificial enzyme, utilization of immune system has been the most effective strategy. However, acceleration of the reaction in terms ofk cat/k uncat by catalytic antibodies is modest when compared with that of natural enzymes (34Kirby A.J. Angew. Chem. Int. Ed. Engl. 1996; 35: 707-724Crossref Scopus (436) Google Scholar). Catalytic antibodies are designed to bind to a transition state analog and speed up the reaction by decreasing the energy of the transition state. Although transition state stabilization is one essential role of an enzyme, it is not the only role, and this may be one of the reasons that catalytic antibodies are less effective than the natural enzymes. Because the enzyme reaction can be divided into multiple steps, the function of the enzyme should also be considered as the assembly of multiple elements. Thus, it is very important for the elucidation of the architecture of enzyme function to understand the polypeptide chain as an assembly of continuous peptide segments, or functional elements. To understand the distribution of the units within the peptide chain, manipulation of main chain connectivity, but not of side chains, would be helpful. In this paper, we described the utility of circular permutation analysis for the discrimination of functional elements.Although successful constructions of a number of circularly permuted proteins have been reported, cleavage of one of the peptide bonds in a protein will not always generate an active and/or stable variant. In our study, the variants from cpL8 to cpI14 could not be obtained because the corresponding E. coli transformants were TMP-sensitive. This result suggests that the chain connectivity of this region is necessary for the formation of stable tertiary structure, namely, folding of the enzyme (Fig. 1, case 1). On the other hand, peptide bond cleavage at each position between Gly-15 and Asp-27 yielded active and stable variants, cpG15∼cpD27. Successful construction of these variants enabled the systematic analysis of the chain connectivity at the M20 loop.Comparison of the activities of CP variants (Fig. 2) revealed three boundaries that divide the peptide chain around the M20 loop into four regions (region 1, ∼Met-16; region 2, Met-16∼Met-20; region 3, Met-20∼Leu-24; region 4, Leu-24∼). These boundaries were also suggested, but in a more subtle fashion, by the alanine (and glycine) scanning experiments (Fig. 6). The variants permuted at region 1 were not studied, except for cpG15, because of poor accumulation in the transformed E. coli cells. For cpG15, the far UV CD spectrum suggested that the main chain fold was not similar to the wild-type (Fig. 3), although Gly-15 is located in the middle of the M20 loop. Thus, region 1 was characterized as a region important for tertiary structure formation of the enzyme (i.e. a folding element) rather than as a functional element. The other regions, regions 2, 3, and 4, were clearly assigned as functional elements around the M20 loop. Although deuterium isotope effect measurements did not identify the catalytic step contributed by the functional elements, studies on the equilibrium dissociation constants suggested that these regions play distinct roles in ligand binding (Table II). Specifically, region 2 was shown to be involved in binding of NADPH and NADP+, whereas region 4 was involved in binding of DHF and THF. These tendencies were in agreement with the crystallographic data available from Protein Data Base files 1rx1, 1rx9, 1rx7, and 1rx5, which represent wild-type DHFR complexed with NADPH, NADP+, folate, and 5,10-dideazatetrahydrofolate, respectively. Although the characteristics of the regions in ligand binding were similar to those obtained by crystallographic data, experimental materials in which chain connectivity at various positions are systematically perturbed are provided solely by the use of circular permutation. Therefore, circular permutation analysis gives an experimental approach to elucidate the role of functional element.Detailed characteristics of the conformation around the M20 loop also supported the division of the loop into multiple elements. The conformation of the M20 loop in the crystals has been classified into three types: closed, occluded, and open conformations (22Sawaya M.R. Kraut J. Biochemistry. 1997; 36: 586-603Crossref PubMed Scopus (604) Google Scholar). In the closed conformation, Met-16∼Ala-19 forms an antiparallel sheet and type III′ hairpin. In the occluded conformation, Glu-17∼Met-20 form a 310 helix. In the open conformation, although the conformation is irregular, the side chains of Met-16 and Met-20 form stabilizing hydrophobic contacts within the loop. The above regions all coincided with region 2. From the coordinates of α carbons forming the M20 loop in these conformations, the extent of the movements of each residues could be compared (Fig. 5). If the M20 loop moves as a single string that cannot be further divided, the shape of the plot of movement versus residue number should consist of single peak. However, the shape of the plot consisted of two peaks around Glu-17 and Pro-21 (Fig. 5). This means that regions of the peptide chain around Glu-17 and Pro-21 move separately. These segments, which were distinct in terms of movement, correspond to two functional elements, region 2 and region 3. The finding that the unit of movement and the unit of functioning peptide (functional element) coincided supports the idea that movement of the peptide backbone is a key feature of enzyme function.There have been many attempts to understand a protein as being composed of multiple segments. From the tertiary structure of a protein, domains and secondary structural units can be easily recognized. Additionally, compact structural units called “modules” are found by an algorithm using the coordinates of Cα atoms (35Go M. Nature. 1981; 291: 90-92Crossref PubMed Scopus (334) Google Scholar, 36Go M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1964-1968Crossref PubMed Scopus (138) Google Scholar). In addition to the computational methods, an experimental method to identify the segments of the protein is the modification on the main chain connectivity. The simplest way to modify main chain connectivity is to dissect the polypeptide at a certain position by fragmentation of the coding gene. When the dissection occurs at the boundary of proper segments such as domains (37Toyama H. Tanizawa K. Yoshimura T. Asano S. Lim Y.H. Esaki N. Soda K. J. Biol. Chem. 1991; 266: 13634-13639Abstract Full Text PDF PubMed Google Scholar, 38Burbaum J.J. Schimmel P. Biochemistry. 1991; 30: 319-324Crossref PubMed Scopus (54) Google Scholar), modules (39Eder J. Kirschner K. Biochemistry. 1992; 31: 3617-3625Crossref PubMed Scopus (75) Google Scholar), and exon-coded segments (40Bertolaet B.L. Knowles J.R. Biochemistry. 1995; 34: 5736-5743Crossref PubMed Scopus (35) Google Scholar), the peptide fragments may associate to give an active complex molecule as high as the activity of the parental protein. Circular permutation is a useful alternative to cleaving main chain connectivity because it does not cause fragmentation of the polypeptide chain. Using this method, we detected multiple functional elements in the M20 loop of E. coli DHFR. Similarly, by examining whether the CP variant can fold stably, peptide regions that play important roles in stable folding (folding elements) can be distinguished from the other regions that do not. Peptide bond cleavage within folding elements will prevent the folding to stable tertiary structure, whereas cleavage outside the folding elements will not. Based on this idea, the M20 loop could be divided not only into functional elements but also into folding elements. Among the regions divided in this study, region 1 can be assigned as a folding element, whereas, region 4, which does not belong to a folding element, forms α helix B. This observation suggests that folding elements are not always characterized as a unit of secondary structure. Systematic construction of the CP variants at all the positions of DHFR (namely, 158 sites) has been completed and provided an overview of folding elements in the enzyme (41Iwakura M. Nakamura T. Protein Sci. 1998; 7 Suppl. 1: 85Google Scholar). Because circular permutation has been reported for many proteins other than DHFR (3Luger K. Hommel U. Herold M. Hofsteenge J. Kirschner K. Science. 1989; 243: 206-210Crossref PubMed Scopus (191) Google Scholar,7Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11980-11984Crossref PubMed Scopus (47) Google Scholar, 8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar, 9Horlick R.A. George H.J. Cooke G.M. Tritch R.J. Newton R.C. Dwivedi A. Lischwe M. Salemme F.R. Weber P.C. Horuk R. Protein Eng. 1992; 5: 427-431Crossref PubMed Scopus (32) Google Scholar, 10Zhang T. Bertelsen E. Benvegnu D. Alber T. Biochemistry. 1993; 32: 12311-12318Crossref PubMed Scopus (82) Google Scholar, 11Hahn M. Piotukh K. Borriss P. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10417Crossref PubMed Scopus (77) Google Scholar, 12Mullins L.S. Wesseling K. Kuo J.M. Garrett J.B. Raushel F.M. J. Amer. Chem. Soc. 1994; 116: 5529-5533Crossref Scopus (19) Google Scholar, 13Viguera A.R. Blanco F.J. Serrano L. J. Mol. Biol. 1995; 247: 670-681Crossref PubMed Scopus (126) Google Scholar, 14Kreitman R.J. Puri R.K. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6889-6893Crossref PubMed Scopus (105) Google Scholar, 15Vignais M.L. Corbier C. Mulliert G. Branlant C. Branlant G. Protein Sci. 1995; 4: 994-1000Crossref PubMed Scopus (18) Google Scholar, 16Kommar A.A. Jaenicke R. FEBS Lett. 1995; 376: 195-198Crossref PubMed Scopus (39) Google Scholar), the approach used in this study should be generally applicable to many proteins. The active site of an enzyme contains amino acid residues involved in enzyme function. Some residues in the active site bind to the substrate or cofactor, and others are involved in the catalysis itself. In addition, residues away from the active site sometimes promote the catalytic reaction through intramolecular interactions (1Miller G.P. Benkovic S.J. Biochemistry. 1998; 37: 6327-6335Crossref PubMed Scopus (64) Google Scholar, 2Miller G.P. Benkovic S.J. Biochemistry. 1998; 37: 6336-6342Crossref PubMed Scopus (59) Google Scholar). Enzyme-catalyzed reactions proceed, in general, through multiple steps, including binding of the substrate, catalysis, and release of product. At each step, certain amino acid residues play critical roles. However, an isolated collection of these directly functioning residues is not sufficient to obtain catalytic activity. Amino acid residues should be connected covalently to make up a polypeptide chain with a specific amino acid sequence that determines a proper tertiary structure. The functioning residues on the peptide backbone are then arranged to give the proper configuration for effective catalytic activity. Because enzymes cleaved at certain sites have been demonstrated to show catalytic activity as high as that of uncleaved enzyme (3Luger K. Hommel U. Herold M. Hofsteenge J. Kirschner K. Science. 1989; 243: 206-210Crossref PubMed Scopus (191) Google Scholar, 4Buchwalder A. Szadkowski H. Kirschner K. Biochemistry. 1992; 31: 1621-1630Crossref PubMed Scopus (74) Google Scholar, 5Protasova N.Y. Kireeva M.L. Murzina N.V. Murzin A.G. Uverski V.N. Gryaznova O.I. Gudkov A.T. Protein Eng. 1994; 7: 1373-1377Crossref PubMed Scopus (43) Google Scholar, 6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar, 7Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11980-11984Crossref PubMed Scopus (47) Google Scholar, 8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar, 9Horlick R.A. George H.J. Cooke G.M. Tritch R.J. Newton R.C. Dwivedi A. Lischwe M. Salemme F.R. Weber P.C. Horuk R. Protein Eng. 1992; 5: 427-431Crossref PubMed Scopus (32) Google Scholar, 10Zhang T. Bertelsen E. Benvegnu D. Alber T. Biochemistry. 1993; 32: 12311-12318Crossref PubMed Scopus (82) Google Scholar, 11Hahn M. Piotukh K. Borriss P. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10417Crossref PubMed Scopus (77) Google Scholar, 12Mullins L.S. Wesseling K. Kuo J.M. Garrett J.B. Raushel F.M. J. Amer. Chem. Soc. 1994; 116: 5529-5533Crossref Scopus (19) Google Scholar, 13Viguera A.R. Blanco F.J. Serrano L. J. Mol. Biol. 1995; 247: 670-681Crossref PubMed Scopus (126) Google Scholar, 14Kreitman R.J. Puri R.K. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6889-6893Crossref PubMed Scopus (105" @default.
• W2027747381 created "2016-06-24" @default.
• W2027747381 creator A5063586448 @default.
• W2027747381 creator A5085191264 @default.
• W2027747381 date "1999-07-01" @default.
• W2027747381 modified "2023-10-16" @default.
• W2027747381 title "Circular Permutation Analysis as a Method for Distinction of Functional Elements in the M20 Loop of Escherichia coliDihydrofolate Reductase" @default.
• W2027747381 cites W1601844248 @default.
• W2027747381 cites W1627023735 @default.
• W2027747381 cites W1850507599 @default.
• W2027747381 cites W1871390403 @default.
• W2027747381 cites W1970692052 @default.
• W2027747381 cites W1978554170 @default.
• W2027747381 cites W1988901563 @default.
• W2027747381 cites W1996143895 @default.
• W2027747381 cites W1996206762 @default.
• W2027747381 cites W2005194798 @default.
• W2027747381 cites W2012798760 @default.
• W2027747381 cites W2012983255 @default.
• W2027747381 cites W2020691090 @default.
• W2027747381 cites W2025960565 @default.
• W2027747381 cites W2029967359 @default.
• W2027747381 cites W2030899314 @default.
• W2027747381 cites W2033343909 @default.
• W2027747381 cites W2037342239 @default.
• W2027747381 cites W2038263678 @default.
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