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• W1995469488 abstract "Aromatic amino acid aminotransferase (AroAT) and aspartate aminotransferase (AspAT) are known as dual-substrate enzymes, which can bind acidic and hydrophobic substrates in the same pocket (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem.(Tokyo) 122, 55–63). In order to elucidate the mechanism of hydrophobic substrate recognition, kinetic and thermodynamic analyses using substrates with different hydrophobicities were performed. They revealed that 1) amino acid substrate specificity (k max/Kd) depended on the affinity for the substrate (1/Kd) and 2) binding of the hydrophobic side chain was enthalpy-driven, suggesting that van der Waals interactions between the substrate-binding pocket and hydrophobic substrate predominated. Three-dimensional structures of AspAT and AroAT bound to α-aminoheptanoic acid were built using the homology modeling method. A molecular dynamic simulation study suggested that the outward-facing position of the Arg292side chain was the preferred state to a greater extent in AroAT than AspAT, which would make the hydrophobic substrate bound state of the former more stable. Furthermore, AroAT appeared to have a more flexible conformation than AspAT. Such flexibility would be expected to reduce the energetic cost of conformational rearrangement induced by substrate binding. These two mechanisms (positional preference of Arg and flexible conformation) may account for the high activity of AroAT toward hydrophobic substrates. Aromatic amino acid aminotransferase (AroAT) and aspartate aminotransferase (AspAT) are known as dual-substrate enzymes, which can bind acidic and hydrophobic substrates in the same pocket (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem.(Tokyo) 122, 55–63). In order to elucidate the mechanism of hydrophobic substrate recognition, kinetic and thermodynamic analyses using substrates with different hydrophobicities were performed. They revealed that 1) amino acid substrate specificity (k max/Kd) depended on the affinity for the substrate (1/Kd) and 2) binding of the hydrophobic side chain was enthalpy-driven, suggesting that van der Waals interactions between the substrate-binding pocket and hydrophobic substrate predominated. Three-dimensional structures of AspAT and AroAT bound to α-aminoheptanoic acid were built using the homology modeling method. A molecular dynamic simulation study suggested that the outward-facing position of the Arg292side chain was the preferred state to a greater extent in AroAT than AspAT, which would make the hydrophobic substrate bound state of the former more stable. Furthermore, AroAT appeared to have a more flexible conformation than AspAT. Such flexibility would be expected to reduce the energetic cost of conformational rearrangement induced by substrate binding. These two mechanisms (positional preference of Arg and flexible conformation) may account for the high activity of AroAT toward hydrophobic substrates. Many enzymes show restricted specificities for single chemical types of substrate (1Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York1985: 389-452Google Scholar), but some have evolved binding pockets with dual specificities for different chemical groups (aminotransferases (Refs. 2Kuramitsu S. Hiromi K. Hayashi H. Morino Y. Kagamiyama H. Biochemistry. 1990; 29: 5469-5476Crossref PubMed Scopus (166) Google Scholarand 3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar) and cysteine protease cruzain (Ref. 4Gillmor S.A. Craik C.S. Fletterick R.J. Protein Sci. 1997; 6: 1603-1611Crossref PubMed Scopus (161) Google Scholar)). Escherichia coli aromatic amino acid aminotransferase (AroAT) 1The abbreviations used are: AroAT, aromatic amino acid aminotransferase; AspAT, aspartate aminotransferase; cAspAT, cytosolic AspAT; mAspAT, mitochondrial AspAT; sCn, α-aliphatic amino acid with a straight side chain bearingn carbon atoms; sCnI, aliphatic acid with a straight side chain bearing n carbon atoms; ksCn, aliphatic α-keto acid with a straight side chain bearing ncarbon atoms; MD, molecular dynamic; PPY,N,5′-phosphopyridoxyl-l-tyrosine; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate. 1The abbreviations used are: AroAT, aromatic amino acid aminotransferase; AspAT, aspartate aminotransferase; cAspAT, cytosolic AspAT; mAspAT, mitochondrial AspAT; sCn, α-aliphatic amino acid with a straight side chain bearingn carbon atoms; sCnI, aliphatic acid with a straight side chain bearing n carbon atoms; ksCn, aliphatic α-keto acid with a straight side chain bearing ncarbon atoms; MD, molecular dynamic; PPY,N,5′-phosphopyridoxyl-l-tyrosine; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate. and aspartate aminotransferase (AspAT) are unique in being active toward two entirely different kinds of substrate (acidic and hydrophobic). These enzymes recognize a carboxyl group of an acidic substrate with side chain of arginine residue and recognize hydrophobic substrates in proportion to their hydrophobicities (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar). These two kinds of substrates are accommodated in the one binding pocket of the enzyme. Structural determination and protein engineering techniques have made it relatively easy to define the key residues that recognize the polar and charged groups of a substrate. However, the mechanism responsible for hydrophobic substrate specificity is more complicated, because the complexity of interactions within the substrate binding pocket and multiple protein configurations must be taken into consideration. In many cases, a number of residues contribute to hydrophobic ligand specificity. There are also situations when the residues determining specificity do not interact directly with the substrate. Generally, hydrophobic side chains are well packed in the protein cores and a hydrophobic effect is one of the major factors involved in substrate recognition and protein folding (5Creighton T.E. Proteins. 2nd Ed. W. H. Freeman and Co., New York1993: 217-238Google Scholar). So far, three strategies designed to estimate the extent of hydrophobic interaction have been reported. The first involves measuring enzyme affinities for a series of hydrophobic ligands (6Hansch C. Coats E. J. Pharm. Sci. 1970; 59: 731-743Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 7Dorovska-Taran V. Momtcheva R. Gulubova N. Martinek K. Biochim. Biophys. Acta. 1982; 702: 37-53Crossref PubMed Scopus (17) Google Scholar, 8Klyosov A.A. Biochemistry. 1996; 35: 4457-4467Crossref PubMed Scopus (126) Google Scholar, 9Lu W. Apostol I. Qasim M.A. Warne N. Wynn R. Zhang W.L. Anderson S. Chiang Y.W. Ogin E. Rothberg I. Ryan K. Laskowski Jr., M. J. Mol. Biol. 1997; 266: 441-461Crossref PubMed Scopus (160) Google Scholar), and detailed thermodynamic analyses of nonpolar interactions based on ligand binding have been performed in T4 lysozyme (10Morton A. Baase W.A. Matthews B.W. Biochemistry. 1995; 34: 8564-8575Crossref PubMed Scopus (157) Google Scholar), acyl-coenzyme A-binding protein (11Færgeman N.J. Sigurskjold B.W. Kragelund B.B. Andersen K.V. Knudsen J. Biochemistry. 1996; 35: 14118-14126Crossref PubMed Scopus (123) Google Scholar), and cytochrome P450cam (12Helms V. Deprez E. Gill E. Barret C. Hoa G.H.B. Wade R.C. Biochemistry. 1996; 35: 1485-1499Crossref PubMed Scopus (48) Google Scholar). The second involves measuring the effects of a series of mutations on protein stability. Several studies have shown that replacing a residue facing the cavity with a more hydrophobic residue makes proteins more stable (13Jackson S.E. Moracci M. elMasry N. Johnson C.M. Fersht A.R. Biochemistry. 1993; 32: 11259-11269Crossref PubMed Scopus (278) Google Scholar, 14Yutani K. Ogasahara K. Tsujita T. Sugino Y. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4441-4444Crossref PubMed Scopus (253) Google Scholar, 15Shortle D. Stites W.E. Meeker A.K. Biochemistry. 1990; 29: 8033-8041Crossref PubMed Scopus (390) Google Scholar, 16Sandberg W.S. Terwilliger T.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1706-1710Crossref PubMed Scopus (117) Google Scholar, 17Serrano L. Kellis Jr., J.T. Cann P. Matouschek A. Fersht A.R. J. Mol. Biol. 1992; 224: 783-804Crossref PubMed Scopus (389) Google Scholar, 18Eriksson A.E. Baase W.A. Matthews B.W. J. Mol. Biol. 1993; 229: 747-769Crossref PubMed Scopus (196) Google Scholar, 19Akasako A. Haruki M. Oobatake M. Kanaya S. J. Biol. Chem. 1997; 272: 18686-18693Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The third involves the use of a series of solvents with different hydrophobicities as the reaction media (20Wangikar P.P. Rich J.O. Clark D.S. Dordick J.S. Biochemistry. 1995; 34: 12302-12310Crossref PubMed Scopus (33) Google Scholar). The results obtained suggest that the effect of a hydrophobic interaction varies according to the environment around the site (hydrophobicity, packing density, conformational flexibility, and presence of water). Aminotransferases catalyze the reversible transamination reaction between an α-amino acid and an α-keto acid, which is accompanied by interconversion of the cofactor between pyridoxal 5′-phosphate (PLP) and pyridoxamine 5′-phosphate (PMP) (21Kiick D.M. Cook P.F. Biochemistry. 1983; 22: 375-382Crossref PubMed Scopus (91) Google Scholar, 22Jenkins W.T. Fonda M.L. Christen P. Metzler D.E. Transaminases. John Wiley & Sons, New York1985: 216-234Google Scholar). Amino acid+EL⇄keto acid+EM REACTION1 E L and E M denote the PLP and PMP forms of the enzyme, respectively. Several kinds of aminotransferases with different substrate specificities are known, and they have been classified into four families on the basis of their amino acid sequence homologies (23Mehta P.K. Hale T.I. Christen P. Eur. J. Biochem. 1993; 214: 549-561Crossref PubMed Scopus (354) Google Scholar, 24Jensen R.A. Gu W. J. Bacteriol. 1996; 178: 2161-2171Crossref PubMed Scopus (143) Google Scholar). E. coli AroAT shows high activity toward hydrophobic and acidic substrates (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar, 25Hayashi H. Inoue K. Nagata T. Kuramitsu S. Kagamiyama H. Biochemistry. 1993; 32: 12229-12239Crossref PubMed Scopus (92) Google Scholar). AroAT is quite similar in many respects to AspAT, which is the most extensively investigated aminotransferase (26John R.A. Biochim. Biophys. Acta. 1995; 1248: 81-96Crossref PubMed Scopus (326) Google Scholar,27Hayashi H. J. Biochem. ( Tokyo ). 1995; 118: 463-473Crossref PubMed Scopus (147) Google Scholar). The three-dimensional structures of AspATs from various sources have been determined by x-ray crystallography (28Smith D.L. Almo S.C. Toney M.D. Ringe D. Biochemistry. 1989; 28: 8161-8167Crossref PubMed Scopus (103) Google Scholar, 29Okamoto A. Higuchi T. Hirotsu K. Kuramitsu S. Kagamiyama H. J. Biochem. ( Tokyo ). 1994; 116: 95-107Crossref PubMed Scopus (143) Google Scholar, 30Rhee S. Silva M.M. Hyde C.C. Rogers P.H. Metzler C.M. Metzler D.E. Arnone A. J. Biol. Chem. 1997; 272: 17293-17302Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). E. coli AroAT (31Kuramitsu S. Inoue K. Ogawa T. Ogawa H. Kagamiyama H. Biochem. Biophys. Res. Commun. 1985; 133: 134-139Crossref PubMed Scopus (44) Google Scholar) and AspAT (32Kuramitsu S. Okuno S. Ogawa T. Ogawa H. Kagamiyama H. J. Biochem. ( Tokyo ). 1985; 97: 1259-1262Crossref PubMed Scopus (53) Google Scholar) show amino acid sequence identities of 43%, and the residues in their catalytic sites are almost fully conserved. Both enzymes have a dimer structure consisting of identical 44-kDa subunits, and their circular dichroism spectra in the far UV region are essentially identical (25Hayashi H. Inoue K. Nagata T. Kuramitsu S. Kagamiyama H. Biochemistry. 1993; 32: 12229-12239Crossref PubMed Scopus (92) Google Scholar). The absorption spectra over 300 nm and pKa values of the PLP-Lys Schiff base are also very similar between AspAT and AroAT, which reflect the microenvironment of the active site. Therefore, it seems reasonable to assume that they have common three-dimensional structures and catalytic mechanisms. The characteristic difference between AspAT and AroAT is the their activity toward hydrophobic substrates; in effect, AroAT shows about 10,000 times higher activity toward phenylalanine than AspAT. Furthermore, the chimera constructed from AspAT and AroAT shows hydrophobic substrate specificities between those of the two parent enzymes (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar, 33Miyazawa K. Kawaguchi S. Okamoto A. Kato R. Ogawa T. Kuramitsu S. J. Biochem. ( Tokyo ). 1994; 115: 568-577Crossref PubMed Scopus (19) Google Scholar). Because the substrate-binding pockets of these three enzymes are surrounded by the same set of residues (residues inred in Fig. 1), the mechanism of hydrophobic substrate recognition by aminotransferases has generated considerable interest. Previous studies (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar) have revealed that the size of the substrate side chain is a major factor that determines the enzymatic activity; the free energy stabilizations of AroAT toward a series of hydrophobic substrates are twice those of AspAT. In particular, AroAT shows constant activity toward the hydrophobic unit of a substrate, irrespective of its shape, suggesting that conformational flexibility or plasticity may be related to the hydrophobic substrate specificity of this enzyme (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar). Model structures for AroAT have been proposed (34Seville M. Vincent M.G. Hahn K. Biochemistry. 1988; 27: 8344-8349Crossref PubMed Scopus (29) Google Scholar, 35Jäger J. Solmajer T. Jansonius J.N. FEBS Lett. 1992; 306: 234-238Crossref PubMed Scopus (14) Google Scholar), and subsequently, the structure of an AspAT hexamutant (V39L, K41Y, T47I, N69L, T109S, and N297S) 2The amino acid residues are numbered according to the sequence of pig cytosolic aspartate aminotransferase (68Mehta P.K. Hale T.I. Christen P. Eur. J. Biochem. 1989; 186: 249-253Crossref PubMed Scopus (124) Google Scholar). 2The amino acid residues are numbered according to the sequence of pig cytosolic aspartate aminotransferase (68Mehta P.K. Hale T.I. Christen P. Eur. J. Biochem. 1989; 186: 249-253Crossref PubMed Scopus (124) Google Scholar). with higher aromatic activity was determined by x-ray crystallography (36Malashkevich V.N. Onuffer J.J. Kirsch J.F. Jansonius J.N. Nat. Struct. Biol. 1995; 2: 548-553Crossref PubMed Scopus (67) Google Scholar). The hexamutant structure is thought to be a better template for AroAT model building than those used previously. In this study, we re-built an AroAT model in its complexed form with α-aminoheptanoic acid that we expected to be helpful for understanding the hydrophobic substrate specificity of AroAT. Recently, the dynamic natures of proteins have been evaluated using a variety of methods. Increasing instances of multiple conformations of substrates and proteins, even in their crystallographic structures, have been found as the result of advances in the various techniques. Protein dynamics, as well as protein structures, are thought to be hierarchical (37Karplus M. McCammon J.A. Annu. Rev. Biochem. 1983; 52: 263-300Crossref PubMed Scopus (566) Google Scholar). Not only large scale conformational changes (movements of domains and/or segments), but also relatively small fluctuations, must be biologically important. In this paper, we discuss protein fluctuation, that may be related to van der Waals interactions within the AroAT enzyme and between this enzyme and its substrates. Molecular dynamic (MD) simulation has become a powerful tool for analyzing the dynamic natures of macromolecules. We used this technique to investigate the flexibility of AroAT and performed thermodynamic analysis to examine further the effects of hydrophobicity on substrate binding. The plasmid pUC19GpY (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar) overproducesE. coli AroAT coded by the tyrB gene in theE. coli TY103 strain (38Yano T. Kuramitsu S. Tanase S. Morino Y. Hiromi K. Kagamiyama H. J. Biol. Chem. 1991; 266: 6079-6085Abstract Full Text PDF PubMed Google Scholar), which was derived from the JM103 strain (39Messing J. Crea R. Seeburg P.H. Nucleic Acids Res. 1981; 9: 309-321Crossref PubMed Scopus (1550) Google Scholar), by disrupting the aspC, tyrB, andrecA genes. Cells carrying this plasmid were grown overnight at 37 °C, harvested by centrifugation, frozen, and stored at −20 °C. The enzyme was purified and its concentration determined using the protocols described previously (33Miyazawa K. Kawaguchi S. Okamoto A. Kato R. Ogawa T. Kuramitsu S. J. Biochem. ( Tokyo ). 1994; 115: 568-577Crossref PubMed Scopus (19) Google Scholar). After the addition of an amino acid substrate to the PLP form of AroAT, the 360-nm absorption band shifts to 330 nm, which means the PLP form has converted to the PMP form. The converse spectral change is observed with the reaction between a keto acid and the PMP form of AroAT. The substrate concentrations used were high enough to convert the enzyme between the PLP and PMP forms. Under single-turnover conditions, the absorption changes at 360 nm were monitored using a stopped-flow spectrophotometer (SX-17MV, Applied Photophysics). The dead time was 2.0 ms under the conditions used (7 kg·cm−2). About 10 progress curves for each substrate concentration were recorded and curve-fitting to a single exponential time course was performed using the program provided with SX-17MV. The kinetic parameters were determined using the following model (Reaction 2) and Equation 1. EL+S⇌KdE·S→kmaxES‡ REACTION2 kapp=kmax[S]/(Kd+[S])Equation 1 k app is the apparent rate constant at a given substrate concentration, k max is the maximum rate constant, and Kd is the dissociation constant. For slow kinetic experiments, a Hitachi U-3000 spectrophotometer was employed. When the k app value was directly proportional to the substrate concentration, Equation 2, instead of Equation 1, was used to determine the catalytic efficiency,k max/Kd(2). kapp=(kmax/Kd)[S]Equation 2 The reaction conditions were 10 μm AroAT, 1–50 mm substrate, 100 mm KCl, 50 mmHEPES-KOH, pH 8.0, and 15–35 °C. Aliphatic acid competitive inhibitors bind noncovalently to AroAT and form Michaelis complexes. Upon complex formation, the pKa of the internal aldimine, which forms between Nε of Lys258 and C4′ of PLP, increases from 6.8 to over 8.0 (40Jenkins W.T. D'Ari L. J. Biol. Chem. 1966; 241: 5667-5674Abstract Full Text PDF PubMed Google Scholar, 41Iwasaki M. Hayashi H. Kagamiyama H. J. Biochem. ( Tokyo ). 1994; 115: 156-161Crossref PubMed Scopus (18) Google Scholar). Therefore, the conversion from the unprotonated (360 nm) and protonated (430 nm) species can be followed spectrophotometrically. The spectra of the PLP form of AroAT with various concentration of aliphatic acids were recorded using a spectrophotometer (Hitachi U-3000). The reaction conditions were 100 mm KCl, 50 mm HEPES-KOH, pH 8.0, 25 °C, and the protein concentration was about 10 μm. The binding of aliphatic acid inhibitors to the PMP form of AroAT was monitored using a spectropolarimeter (Jasco J-720). The Kd values for the inhibitors were obtained by fitting the absorbances at 430 (PLP form) or 330 nm (PMP form) to theoretical curves using the Igor Pro version 3.01 application (WaveMetrics, Inc.). The free energy change for the fast binding step (ΔG s) was obtained using the equation −RTln(1/Kd), where Ris the gas constant (1.99 cal/K·mol) and T is the absolute temperature, and the enthalpy change (ΔH s) was derived from a van't Hoff equation. δln1Kd/δT=ΔHsRT2Equation 3 This equation is often integrated under the assumption that ΔCp = 0. ln1Kd=lnA−ΔHsRTEquation 4 ΔH s in Equation 4 is the so-called van't Hoff enthalpy. The reactions between the PMP form of the enzyme and keto acid substrates were analyzed using Equation 4. When it is supposed that the enthalpy depends linearly on temperature, the enthalpy change is expressed as shown in Equation 5. ΔHs=ΔHr+ΔCp(T−Tr)Equation 5 ΔH r is the enthalpy change at temperature T r, and ΔCp is the non-zero heat capacity change. Therefore, integration of the van't Hoff equation (Equation 3) yields Equation 6. ln1Kd=ln1Kr+ΔCpR−(1+lnTr)+Tr1T−ln1TEquation 6 K r is the dissociation constant atT r. The reactions between the PLP form of the enzyme and amino acid substrates were analyzed using Equation 6. The entropy change was calculated using the equation ΔG s = ΔH s − TΔS s, and the activation free energy for the rate-determining step (ΔG‡) was calculated using Equation 7. ΔG‡=RTlnkBTh−lnkmaxEquation 7 k B is the Boltzmann constant (1.38 × 10−34 J·K−1), and h is the Planck constant (6.63 × 10−34 J·s). The activation energy (E A) was obtained from an Arrhenius plot of the rate constant versus temperature (ln(k max) versus 1/T), and the slope of this plot yields −E A/R. Thus, the enthalpy change (ΔH‡) is calculated using the equation ΔH‡ = E A −RT, and the activation entropy change (ΔS‡) was calculated using the equation ΔG‡ = ΔH‡ −TΔS‡. The x-ray crystallographic structure of the hexamutant of AspAT (Protein Data Bank code 1AHG; see Ref. 36Malashkevich V.N. Onuffer J.J. Kirsch J.F. Jansonius J.N. Nat. Struct. Biol. 1995; 2: 548-553Crossref PubMed Scopus (67) Google Scholar) complexed with the cofactor-substrate analog N,5′-phosphopyridoxyl-l-tyrosine (PPY) was used as the model for the hydrophobic substrate ligand-enzyme complex. As the crystallographic structure of AspAT complexed with hydrophobic substrate analog has not been resolved, it was built by reconverting the six mutated residues of the AspAT hexamutant. Modeling of E. coli AspAT and AroAT was carried out using the Homology program (Biosym Technologies). AspAT and its hexamutant have the same number of residues, whereas AroAT is one residue longer. Pro64 was interpreted as an insertion residue, when performing homology modeling of AroAT. The three-dimensional structure data base (Protein Data Bank, Release 76) was searched for the structure of residues 58–70, and the most probable loop structure was chosen as an initial structure and spliced into the AroAT structure in order to fill the insertion gap. The tyrosine residue of PPY in the hexamutant was replaced by α-aminoheptanoic acid (sC7), a double bond between N of sC7 and C4′ of PLP and partial double bonds in the aromatic ring of PLP were introduced and a 5-Å shell of water was added to both the AspAT and AroAT structures. The AspAT and AroAT systems contained water molecules found in the template structure (36Malashkevich V.N. Onuffer J.J. Kirsch J.F. Jansonius J.N. Nat. Struct. Biol. 1995; 2: 548-553Crossref PubMed Scopus (67) Google Scholar) and a total of 24,279 and 24,391 atoms, respectively. MD simulation was carried out using the Discover version 2.97 program (Biosym Technologies) with the consistent valence force-field. The dielectric constant was fixed at 1.0, and a 14-Å cutoff was used for the nonbonded interactions with a switching function which smoothly turns off the interaction over a range of 1.5 Å. Minimization was performed according to the strategy used by Kasper et al. (42Kasper P. Sterk M. Christen P. Gehring H. Eur. J. Biochem. 1996; 240: 751-755Crossref PubMed Scopus (9) Google Scholar). Briefly, both the AspAT and AroAT systems were subjected first to 500 energy minimization steps with all the heavy atoms fixed and then to 500 steps with all the atoms free to move, using the steepest-descents algorithm. Subsequently, they were subjected to 2000 steps using the conjugate-gradient algorithm. After minimization, the total energies in AspAT and AroAT were −57 Mcal·mol−1and −53 Mcal·mol−1, respectively, and their respective root mean square derivatives were 0.066 and 0.062 kcal·mol−1·Å−1. The temperatures of both systems reached 300 K during the initial 2 ps of MD simulation. The Discover program assigned initial velocities to the atoms in order to maintain a Maxwell-Boltzmann distribution at a given temperature. The rest of the simulation protocol was run for about 280 and 370 ps for AspAT and AroAT, respectively, at 300 K with a time step of 1 fs, and the molecular coordinates of the molecules were saved every 1 ps. In order to remove the overall translation and rotation of the systems, the backbone N, Cα, and C atoms of 100 conformers (last 100-ps interval of simulation) were superimposed on those of the first conformer within this time interval and each atom position was averaged. The mean square fluctuation was calculated for each Cα atom using Equation 8. 〈ΔR2〉=∑i{(Xi−Xav)2+(Yi−Yav)2+(Zi−Zav)2}/100Equation 8 Xi, Yi, andZi are the Cα coordinates of a given conformer in the 100-ps trajectory; X av,Y av, and Z av are the Cα coordinates of the time-averaged structure. A series of aliphatic substrates of uniform chemical nature was used in this study. The enzymatic activities toward substrates of various hydrophobicities were studied by analyzing the single-turnover reactions (half-reactions) using a stopped-flow apparatus. TableI shows the values ofk max/Kd at 15–35 °C. A temperature increase of 5 °C increased thek max/Kd value for each substrate 2-fold, and the catalytic efficiency increased as the side chain length increased. The energetic contribution of each hydrophobic substrate side chain to the enzymatic activity was evaluated by calculating the ΔG T‡ value using Equation 9 (Scheme FS1) (43Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York1985: 311-346Google Scholar). ΔGT‡=RTlnkBTh−lnkmaxKdEquation 9 The ΔG T‡ value decreased as the carbon number of the substrate increased and, consistent with previous studies (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 55-63Crossref PubMed Scopus (24) Google Scholar, 44Onuffer J.J. Ton B.T. Klement I. Kirsch J.F. Protein Sci. 1995; 4: 1743-1749Crossref PubMed Scopus (29) Google Scholar), the relationship was linear over the 4- to 7-carbon range (Fig. 2). The slopes of the straight lines represented the dependence of the enzymatic activity on the hydrophobic unit of the substrate side chain and were referred to as the hydrophobic “substrate specificities.” Over the temperature range tested (15–35 °C), these slopes were almost identical, −1.6 kcal·mol−1. Contrary to expectation, these results suggest that entropy was not a major determinant of the substrate specificity of AroAT, and they may be attributable to strong enthalpy-entropy compensation.Table IThe kmax/Kd (s−1 ·m−1) values for E. coli AroAT with a series of aliphatic substrates at various temperaturesTemperatureSubstratesC3sC4sC5sC6sC7°C150.802.52676011,000201.65.2611,70023,000253.5121503,30043,000305.2262906,20076,00035115154010,000130,000Conditions: 50 mm HEPES, 100 mm KCl, pH 8.0. sCn, CH3-(CH2)n−3-CH(NH2)-COOH. Open table in a new tab Figure 2Correlation between ΔG T‡ and the substrate carbon numbers. ○, 15 °C; ⋄, 20 °C; □, 25 °C; ▵, 30 °C; ▿, 35 °C. The substrates are a series of aliphatic amino acids with straight side chains. The free energy differences (ΔG T‡) between the unbound enzyme and substrate (E+S) and the transition state (ES‡) was calculated using the equation ΔG T‡ = ΔG s + ΔG‡ =RT(ln(k B T/h) − ln(k max/Kd)). The reaction conditions were 50 mm HEPES buffer containing 100 mm KCl, pH 8.0, at the given temperatures.View Large Image Figure ViewerDownload (PPT) Conditions: 50 mm HEPES, 100 mm KCl, pH 8.0. sCn, CH3-(CH2)n−3-CH(NH2)-COOH. From this analysis, it was not clear how the temperature and substrate hydrophobicity affect the bound and transition states of the reaction. Therefore, separate determinations of k max andKd values were required. Two directions of the half-transamination reaction were defined; the reaction between an amino acid substrate and the PLP form of the enzyme was referred to as the “forward reaction,” and that between a keto acid substrate and the PMP form of the enzyme was referred to as the “reverse reaction.” Table IIshows the kinetic parameters of the forward and reverse reactions at 25 °C.Table IIKinetic parameters of the half-reactions and dissociation constants for E. coli AroATSubstrate or inhibitorEnzymek maxKdΔΔG SΔΔG T‡s−1mmkcal · mol−1kcal · mol−1sC6PLP21058−1.4−1.5sC7PLP2305.1sC7IPLP33−1.2−1.3aCalculated from the difference between thek max/Kd values of amino acids with cognate side chains (3).sC8IPLP4.5ksC5PMP9362−1.1−1.7ksC6PMP38010sC6IPMP72−1.7−1.7bAverage contribution of one methylene group to ΔG T‡ of a series of keto acid substrates (3).sC7IPMP4.4Conditions: 50 mm HEPES, 100 mm KCl, pH 8.0, 25 °C. The abbreviations: sCn, CH3-(CH2)n−3-CH(NH2)-COOH; ksCn, CH3-(CH2)n−3-CO-COOH; sCnI, CH3-(CH2)n−3-CH2-COOH.a Calculated from the difference between thek max/Kd values of amino acids with cognate side chains (3Kawaguchi S. Nobe Y. Yasuoka J. Wakamiya T. Kusumoto S. Kuramitsu S. J. Biochem. ( Tokyo ). 1997; 122: 5" @default.
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• W1995469488 title "Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases" @default.
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