Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases*

Shin-ichi KawaguchiDagger and Seiki Kuramitsu§

From the Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (kmax/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 alpha -aminoheptanoic acid were built using the homology modeling method. A molecular dynamic simulation study suggested that the outward-facing position of the Arg292 side 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Many enzymes show restricted specificities for single chemical types of substrate (1), but some have evolved binding pockets with dual specificities for different chemical groups (aminotransferases (Refs. 2 and 3) and cysteine protease cruzain (Ref. 4)). Escherichia coli aromatic amino acid aminotransferase (AroAT)1 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 (3). 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 (5). 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 (6-9), and detailed thermodynamic analyses of nonpolar interactions based on ligand binding have been performed in T4 lysozyme (10), acyl-coenzyme A-binding protein (11), and cytochrome P450cam (12). 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 (13-19). The third involves the use of a series of solvents with different hydrophobicities as the reaction media (20). 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 alpha -amino acid and an alpha -keto acid, which is accompanied by interconversion of the cofactor between pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) (21, 22).
<UP>Amino acid</UP>+E<SUB><UP>L</UP></SUB> ⇄ <UP>keto acid</UP>+E<SUB><UP>M</UP></SUB>
<UP><SC>Reaction 1</SC></UP>
EL and EM 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 (23, 24).

E. coli AroAT shows high activity toward hydrophobic and acidic substrates (3, 25). AroAT is quite similar in many respects to AspAT, which is the most extensively investigated aminotransferase (26, 27). The three-dimensional structures of AspATs from various sources have been determined by x-ray crystallography (28-30). E. coli AroAT (31) and AspAT (32) 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 (25). 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 (3, 33). Because the substrate-binding pockets of these three enzymes are surrounded by the same set of residues (residues in red in Fig. 1), the mechanism of hydrophobic substrate recognition by aminotransferases has generated considerable interest. Previous studies (3) 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 (3).


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Fig. 1.   Structure of the active site of E. coli AspAT complexed with alpha -methylaspartate (29). The direction of view is from the Calpha atom of the substrate analog to the side chain terminus. a, alpha -methylaspartate is shown in cyan. b, the analog has been omitted from a. Red, side chains conserved in AspAT and AroAT; blue, non-conserved side chains; green, backbone.

Model structures for AroAT have been proposed (34, 35), and subsequently, the structure of an AspAT hexamutant (V39L, K41Y, T47I, N69L, T109S, and N297S)2 with higher aromatic activity was determined by x-ray crystallography (36). 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 alpha -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 (37). 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Enzyme Preparation-- The plasmid pUC19GpY (3) overproduces E. coli AroAT coded by the tyrB gene in the E. coli TY103 strain (38), which was derived from the JM103 strain (39), by disrupting the aspC, tyrB, and recA 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 (33).

Pre-steady-state Kinetic Analysis-- 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.
E<SUB><UP>L</UP></SUB>+<UP>S</UP> <LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB>d</SUB></UL></LIM> E · <UP>S</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>max</UP></SUB></UL></LIM> E<UP>S</UP><SUP>‡</SUP>
<UP><SC>Reaction 2</SC></UP>
k<SUB><UP>app</UP></SUB>=k<SUB><UP>max</UP></SUB> [<UP>S</UP>]/(K<SUB>d</SUB>+[<UP>S</UP>]) (Eq. 1)
kapp is the apparent rate constant at a given substrate concentration, kmax is the maximum rate constant, and Kd is the dissociation constant.

For slow kinetic experiments, a Hitachi U-3000 spectrophotometer was employed. When the kapp value was directly proportional to the substrate concentration, Equation 2, instead of Equation 1, was used to determine the catalytic efficiency, kmax/Kd(2).
k<SUB><UP>app</UP></SUB>=(k<SUB><UP>max</UP></SUB>/K<SUB>d</SUB>)[<UP>S</UP>] (Eq. 2)
The reaction conditions were 10 µM AroAT, 1-50 mM substrate, 100 mM KCl, 50 mM HEPES-KOH, pH 8.0, and 15-35 °C.

Binding of Inhibitors-- Aliphatic acid competitive inhibitors bind noncovalently to AroAT and form Michaelis complexes. Upon complex formation, the pKa of the internal aldimine, which forms between Nepsilon of Lys258 and C4' of PLP, increases from 6.8 to over 8.0 (40, 41). 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.).

Thermodynamic Analysis-- The free energy change for the fast binding step (Delta Gs) was obtained using the equation -RTln(1/Kd), where R is the gas constant (1.99 cal/K·mol) and T is the absolute temperature, and the enthalpy change (Delta Hs) was derived from a van't Hoff equation.
&dgr;<FENCE><UP>ln</UP> <FR><NU>1</NU><DE>K<SUB>d</SUB></DE></FR></FENCE>/&dgr;T=<FR><NU>&Dgr;H<SUB><UP>s</UP></SUB></NU><DE>RT<SUP>2</SUP></DE></FR> (Eq. 3)
This equation is often integrated under the assumption that Delta Cp = 0. 
<UP>ln</UP><FENCE><FR><NU>1</NU><DE>K<SUB>d</SUB></DE></FR></FENCE>=<UP>ln</UP> A−<FR><NU>&Dgr;H<SUB><UP>s</UP></SUB></NU><DE>RT</DE></FR> (Eq. 4)
Delta Hs 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.
&Dgr;H<SUB><UP>s</UP></SUB>=&Dgr;H<SUB><UP>r</UP></SUB>+&Dgr;Cp(T−T<SUB><UP>r</UP></SUB>) (Eq. 5)
Delta Hr is the enthalpy change at temperature Tr, and Delta Cp is the non-zero heat capacity change. Therefore, integration of the van't Hoff equation (Equation 3) yields Equation 6.
   <UP>ln</UP><FENCE><FR><NU>1</NU><DE>K<SUB>d</SUB></DE></FR></FENCE>=<UP>ln</UP><FENCE><FR><NU>1</NU><DE>K<SUB><UP>r</UP></SUB></DE></FR></FENCE>+<FR><NU>&Dgr;Cp</NU><DE>R</DE></FR><FENCE><UP>−</UP>(1+<UP>ln</UP>T<SUB><UP>r</UP></SUB>)+T<SUB><UP>r</UP></SUB><FENCE><FR><NU>1</NU><DE>T</DE></FR></FENCE>−<UP>ln</UP><FENCE><FR><NU>1</NU><DE>T</DE></FR></FENCE></FENCE> (Eq. 6)
Kr is the dissociation constant at Tr. 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 Delta Gs = Delta Hs - TDelta Ss, and the activation free energy for the rate-determining step (Delta GDagger ) was calculated using Equation 7.
&Dgr;G<SUP>‡</SUP>=RT<FENCE><UP>ln</UP><FENCE><FR><NU>k<SUB><UP>B</UP></SUB>T</NU><DE>h</DE></FR></FENCE>−<UP>ln</UP>k<SUB><UP>max</UP></SUB></FENCE> (Eq. 7)
kB 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 (EA) was obtained from an Arrhenius plot of the rate constant versus temperature (ln(kmax) versus 1/T), and the slope of this plot yields -EA/R. Thus, the enthalpy change (Delta HDagger ) is calculated using the equation Delta HDagger  = EA - RT, and the activation entropy change (Delta SDagger ) was calculated using the equation Delta GDagger  = Delta HDagger  - TDelta SDagger .

Homology Modeling and Molecular Dynamic Simulation-- The x-ray crystallographic structure of the hexamutant of AspAT (Protein Data Bank code 1AHG; see Ref. 36) 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 alpha -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 (36) 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. (42). 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-1 and -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, Calpha , 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 Calpha atom using Equation 8.
⟨&Dgr;R<SUP>2</SUP>⟩=<LIM><OP>∑</OP><LL>i</LL></LIM>{(X<SUB>i</SUB>−X<SUB><UP>av</UP></SUB>)<SUP>2</SUP>+(Y<SUB>i</SUB>−Y<SUB><UP>av</UP></SUB>)<SUP>2</SUP>+(Z<SUB>i</SUB>−Z<SUB><UP>av</UP></SUB>)<SUP>2</SUP>}/100 (Eq. 8)
Xi, Yi, and Zi are the Calpha coordinates of a given conformer in the 100-ps trajectory; Xav, Yav, and Zav are the Calpha coordinates of the time-averaged structure.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The Effect of Temperature on Hydrophobic Substrate Specificity

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. Table I shows the values of kmax/Kd at 15-35 °C. A temperature increase of 5 °C increased the kmax/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 Delta GTDagger value using Equation 9 (Scheme 1) (43).
&Dgr;G<SUP>‡</SUP><SUB><UP>T</UP></SUB>=<UP>RT</UP><FENCE><UP>ln</UP><FENCE><FR><NU>k<SUB><UP>B</UP></SUB>T</NU><DE>h</DE></FR></FENCE>−<UP>ln</UP><FENCE><FR><NU>k<SUB><UP>max</UP></SUB></NU><DE>K<SUB><UP>d</UP></SUB></DE></FR></FENCE></FENCE> (Eq. 9)
The Delta GTDagger value decreased as the carbon number of the substrate increased and, consistent with previous studies (3, 44), 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.

                              
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Table I
The kmax/Kd (s-1 · M-1) values for E. coli AroAT with a series of aliphatic substrates at various temperatures
Conditions: 50 mM HEPES, 100 mM KCl, pH 8.0. sCn, CH3-(CH2)n-3-CH(NH2)-COOH.


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Scheme 1.   Free energy diagram of enzymatic reaction (43).


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Fig. 2.   Correlation between Delta GTDagger and the substrate carbon numbers. open circle , 15 °C; diamond , 20 °C; square , 25 °C; triangle , 30 °C; down-triangle, 35 °C. The substrates are a series of aliphatic amino acids with straight side chains. The free energy differences (Delta GTDagger ) between the unbound enzyme and substrate (E+S) and the transition state (ESDagger ) was calculated using the equation Delta GTDagger  = Delta Gs + Delta GDagger  = RT(ln(kBT/h- ln(kmax/Kd)). The reaction conditions were 50 mM HEPES buffer containing 100 mM KCl, pH 8.0, at the given temperatures.

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 kmax and Kd values were required.

Kinetic Analysis of Half-reactions

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 II shows the kinetic parameters of the forward and reverse reactions at 25 °C.

                              
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Table II
Kinetic parameters of the half-reactions and dissociation constants for E. coli AroAT
Conditions: 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.

Forward Reaction-- It was not possible to determine the kinetic parameters for the alanine (sC3), alpha -aminobutyric acid (sC4), or norvaline (sC5) substrate due to their large dissociation constants. The Kd values for norleucine (sC6) and alpha -aminoheptanoic acid (sC7) at 25 °C were 58 and 5.1 mM, respectively. The affinity (1/Kd) of AroAT for the sC7 substrate was 10 times that for the sC6 substrate. In contrast, the kmax values, the first order rate constants, for these two substrates were almost equal. These results suggest that the hydrophobicity of the substrate side chain affected only the bound state and did not change the transition state and that the affinity for the substrate was the major factor that determined the catalytic efficiency in the forward reaction. Consequently, the difference between the free energy changes from the unbound to substrate-bound state (Delta Delta Gs(7-6), -1.4 kcal·mol-1 between sC6 and sC7) was nearly same as the difference between the free energy changes from the unbound to transition state (Delta Delta GTDagger (7-6), -1.5 kcal·mol-1).

The aliphatic acid AroAT inhibitors we used can bind noncovalently to the enzyme and form Michaelis complexes. The dissociation constant for the reaction of heptanoic acid (sC7I) with the PLP form of AroAT was 6 times higher than that of its cognate amino acid substrate, sC7 (Table II). The lower affinity for sC7I was probably attributable to the lack of covalent binding to the cofactor PLP. The energetic contribution of each hydrophobic unit to the affinity, Delta Delta Gs(8-7), was calculated to be -1.2 kcal·mol-1 (between sC7I and sC8I). This value was similar to the Delta Delta GTDagger (8-7) of -1.3 kcal·mol-1 between the sC7 and sC8 substrates, indicating that the hydrophobicities of the inhibitors and amino acid substrates had the same effects on the affinities of the PLP form of AroAT.

The kinetic parameters of the half-reactions were determined at various temperatures, and Table III shows the thermodynamic data for each reaction. The linear relationships between ln(kmax) and 1/T shown by the Arrhenius plots (Fig. 3a) indicate that the reaction mechanisms for sC6 and sC7 substrates did not alter over the temperature range studied and that their Delta HDagger values were similar. Furthermore, their similar kmax values mean that the Delta GDagger values are almost equal. Therefore, it is indicated that the Delta SDagger values of sC6 and sC7 are also similar and that the extra methylene group of the latter did not affect the conformation of the transition state. The van't Hoff plot for the forward reaction yielded a convex curve (Fig. 3c), indicating large heat capacity changes (Delta Cp) had occurred. Negative Delta Cp values may, at least in part, mean that during enzyme-substrate binding, hydrophobic areas of the substrate and enzyme were removed from contact with water. Similarly, a negative Delta Cp value was observed during the binding of octanoic acid (sC8I) to the PLP form of AroAT (data not shown). Murphy and Freire (45) proposed that the contributions of polar and nonpolar interactions to Delta Cp are proportional to the changes in their accessible surface areas (the polar and nonpolar parameters were 0.26 and -0.45 cal·mol-1·K-1·Å-2, respectively, when those areas were removed from contact with water). However, the Delta Cp value for the sC6 substrate was more negative than that for the sC7 substrate, even though the interacting hydrophobic area of sC7 was greater than that of sC6. Therefore, contributions of other factors, i.e. van der Waals interactions, must be considered.

                              
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Table III
Thermodynamic parameters of half-reactions for E. coli AroAT at 25 °C
Conditions: 50 mM HEPES, 100 mM KCl, pH 8.0. Abbreviations: sCn, CH3-(CH2)n-3-CH(NH2)-COOH; ksCn, CH3-(CH2)n-3-CO-COOH. The data shown are means ± S.D.


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Fig. 3.   Arrhenius (a and b) and van't Hoff (c and d) plots of half-reactions. a and c, alpha -Aminohexanoic acid (sC6) (open circle  and solid line); alpha -aminoheptanoic acid (sC7) (square  and broken line). b and d, alpha -ketovaleric acid (ksC5) (open circle  and solid line); alpha -ketocaproic acid (ksC6) (square  and broken line). Straight and curved lines were fitted under the assumption that Delta Cp = 0 and a constant Delta Cp not equal  0, respectively. The reaction conditions were 50 mM HEPES buffer containing 100 mM KCl, pH 8.0, at the given temperatures.

Reverse Reaction-- The catalytic efficiencies of many aminotransferases are greater with keto acid substrates than their cognate amino acid substrates, due to the higher affinities for keto acids. The affinity has a greater effect on the catalytic efficiency than the rate constant (2, 25). The kinetic parameters for alpha -ketovaleric (ksC5) and alpha -ketocaproic (ksC6) acids were determined (Table II). The affinity of AroAT for the ksC6 substrate was 6 times that for the ksC5 substrate and the kmax value for the former was 4 times higher than that for the latter. Both kmax and Kd of the reverse reaction were affected by side chain hydrophobicity, whereas the kmax values for the amino acid substrates we used were not. Consequently, Delta Delta Gs(6-5) (-1.1 kcal·mol-1 between ksC5 and ksC6) was significantly smaller than the cognate Delta Delta GTDagger (6-5) (-1.7 kcal·mol-1) for the reverse reaction. With the PMP form of the enzyme, the difference between the Kd values of sC7I and sC6I was responsible for the Delta Delta Gs(7-6) of -1.7 kcal·mol-1, which was equal to the Delta Delta GTDagger (7-6) (-1.7 kcal·mol-1) value estimated from the substrate specificities for keto acid substrates (see Fig. 1a in Ref. 3). These results suggest that the energy required to stabilize the Michaelis complex was used to stabilize the transition state of the reverse reaction, as well as that of the forward reaction.

Thermodynamic analysis revealed large positive enthalpy changes (3-6 kcal·mol-1) during the substrate-binding step (Fig. 3d). These large Delta Hs value may mean that the solvation energies are higher for keto acids than amino acids. Another explanation is that more water molecules are associated with the PMP form of AroAT than with the PLP form, although fewer water molecules were observed in the crystallographic structure of the PMP form of E. coli AspAT than in that of the PLP form (46). The activation free energies (Delta GDagger ) were 14-15 kcal·mol-1 for all the substrates we used (Table III) and were higher than that for the reaction of aspartate with AspAT by only 1 kcal·mol-1 (2). Therefore, these substrates appear to be almost good substrates for AroAT.

Unlike the forward reaction, the activation free energy of the reverse reaction was altered by the side chain length (Fig. 3b). Furthermore, the contribution of a methylene group in a keto acid substrate to the bound state differed from that of one in an inhibitor (Table II); Delta Delta Gs and Delta Delta GTDagger were both -1.7 kcal·mol-1 for a set of inhibitors, whereas their respective values were -1.1 and -1.7 kcal·mol-1, substantially different, for a set of keto acid substrates. These results suggest that there were some differences between the kinetic bound states in both directions. A possible explanation for this is that an intermediate (E·I), the energy level of which depends on the side chain length, forms and is in rapid equilibrium (Scheme 2), as shown by Reaction 3. 
E<SUB><UP>M</UP></SUB>+<UP>KA</UP> <LIM><OP><ARROW>⇄</ARROW></OP><UL>K<SUB>1</SUB></UL></LIM> E<SUB><UP>M</UP></SUB> · <UP>KA</UP> <LIM><OP><ARROW>⇄</ARROW></OP><UL>K<SUB>2</SUB></UL></LIM> E · I → ‡
<UP><SC>Reaction </SC>3</UP>
EM is the PMP form of AroAT, KA a keto acid, ddager  the transition state of the reaction, K1 the dissociation constant ([EM][KA]/[EM·KA]), and K2 the equilibrium constant ([EM·KA]/[E·I]). In this model, the apparent Kd value for a substrate is expressed by K1·K2/(1+K2). More detailed analysis of the reverse reaction will be reported elsewhere.


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Scheme 2.   Energy profiles of the transamination reactions with substrates with n and n+1 carbons. EL, pyridoxal form of enzyme; AA, amino acid substrate; E·I, intermediate form; EM, pyridoxamine form of enzyme; KA, keto acid substrate.

Energy Profile of the Transamination Reaction

Fig. 4 shows the reaction profiles for the transamination reactions of AroAT with sC6 and ksC6. The free energy level of the unbound PLP form of AroAT plus an amino acid substrate (EL sC6 in Fig. 4) was a little lower by 1.3 kcal·mol-1, than that of the unbound PMP form of AroAT plus a keto acid substrate (EM + ksC6 in Fig. 4). This value agreed well with the value estimated from the equilibrium constant of the half-reaction, which was determined spectrophotometrically (data not shown).


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Fig. 4.   Profiles of the thermodynamic parameters for the half-transamination reactions between AroAT and alpha -aminohexanoic and alpha -ketocaproic acids. Delta G (solid line), Delta H (broken line), and TDelta S (dotted line) are shown. EL, pyridoxal form; EM, pyridoxamine form; sC6, alpha -aminohexanoic acid; ksC6, alpha -ketocaproic acid.

In order to evaluate the hydrophobic effects, the energy difference profiles were evaluated (Table IV). Although, overall, the binding steps were entropy-driven (Fig. 5, a and b), binding of a single methylene group was enthalpy-driven (Fig. 5c). We thought this was because the van der Waals interactions in the substrate-binding pocket were strong and because the substrate side chain was packed well by the surrounding residues of AroAT.

                              
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Table IV
Energetic contribution of a single methylene group in the substrate side chain
The data shown are means ± S.D.


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Fig. 5.   Profiles of the thermodynamic parameters for the forward transamination reaction. a, reaction between alpha -aminohexanoic acid (sC6) and the pyridoxal form of the enzyme. b, reaction between alpha -aminoheptanoic acid (sC7) and the pyridoxal form of the enzyme. c, subtraction of a from b.

Homology Modeling and Molecular Dynamic Simulation

The hexamutant structure (Protein Data Bank code 1AHG) was chosen as a template for model building, because it is the only high resolution structure complexed with a hydrophobic substrate analog, PPY. In this study, the tyrosine residue of this analog was replaced by alpha -aminoheptanoic acid (sC7), because AroAT showed high activity toward not only aromatic substrates, but also aliphatic substrates. Furthermore, sC7 can bind to both AspAT and AroAT without substantial energy cost, and it has been suggested that the substrates larger than sC7 are subject to steric hindrance in the binding pocket of AspAT (3).

After minimizing the initial AroAT and AspAT structures complexed with sC7 molecules, MD simulation for 280 and 370 ps for AspAT and AroAT, respectively, was performed (Fig. 6). The temperature of both systems was 300 ± 2 K, and the kinetic energies were maintained at about 21.5-22.0 Mcal during simulation. The potential energies declined gradually, and that of the AspAT system almost reached equilibrium in 200 ps. Therefore, the 181-280-ps trajectory was analyzed, as described below. Only one water molecule (B128 HOH in 1AHG), which was originally defined in the crystal structure, was apart from the AspAT molecule. A longer simulation time was required for the AroAT system to reach equilibrium, probably because the initial structure of AroAT differed more from that yielded by MD simulation than was the case with AspAT simulation. Therefore, the 271-370-ps trajectory of the AroAT system was analyzed. First, the overall translation and rotation of the molecule were removed by superimposition of the first conformer at the 100-ps interval. The root mean square deviations of the aligned positions were about 0.6 and 0.9 Å for AspAT and AroAT, respectively. Subsequently, the positionally averaged structures were obtained from the 100 aligned conformers.


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Fig. 6.   Time courses of potential energies (a), kinetic energies (b), and temperatures (c) of the AroAT (solid line) and AspAT (dotted line) systems. All data were saved every 1 ps.

The many secondary structural segments defined by the DSSP method (47) were shorter than those in the crystallographic structure (hexamutant, Protein Data Bank code 1AHG; or wild type AspAT with alpha -methylaspartate, Protein Data Bank code 1ART) by one or two residues, particularly for the alpha -helices, although their positions in the primary structure remained constant, and this was probably related to the high mobilities of the atoms at both ends of the secondary structures. The averaged structures were compared with the structures determined experimentally by superimposing their main chain (N, Calpha , and C) atoms, and the root mean square deviations of the two structures in their aligned positions are shown in Table V. The averaged structure of AroAT had higher values than that of AspAT, which may be due to their different loop structures. The structures of both AspAT and AroAT were closer to that of the closed conformation complexed with a substrate analog (1AHG or 1ART) than to that of an open one (Protein Data Bank code 1ARS) (Fig. 7). When wild type AspAT bound to the substrate analog, the small domain (5-52 and 364-409 residues) is observed to move 5° toward the large domain (53-363 residues) and the dihedral angles of three residues (36, 37, and 38) changed (29). The dihedral angles of these residues in the averaged structures were similar to those of wild type AspAT in the closed conformation, except that Gly36 in subunit A of the averaged AspAT adopted a dihedral angle similar to that of the open conformation. These results suggest that even wild type AspAT in its closed conformation complexed with a non-dicarboxylate substrate (hydrophobic substrate) was sufficiently stable to catalyze the transamination reaction. Two Pro residues (138 and 195) maintained their cis-conformations during simulation, and these were the same as the conformations observed in the crystallographic structures.

                              
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Table V
Root mean square deviations (Å) of the backbone (N, Calpha , and C) atoms of two structures


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Fig. 7.   Stereo drawings of the backbone structures. The averaged structure of AroAT (magenta), crystallographic structure of the open form of wild type AspAT (yellow), and crystallographic structure of the closed form of wild type AspAT (cyan) are superimposed on the averaged structure of AspAT (white).

The structures of the substrate binding sites of AspAT and AroAT were fairly similar with the main differences around the Arg292 residue. In subunit A of AspAT, the side chain of Arg292 had turned toward the interior of the active site and its guanidino group had formed hydrogen bonds with two water molecules, a position like that of the omega -carboxyl group of the natural substrate for AspAT (Fig. 8a). The side chains of Arg292 in the B subunit of AspAT (Fig. 8b) and both AroAT subunits (Fig. 8, c and d) were at the edge of the binding site, where all four Arg292 residues existed when simulation was started. The movement of the Arg292 side chain from the edge to the interior of subunit A occurred 4-7 ps after starting the MD simulation. At the beginning of simulation, the total energy of the system was higher than that during the equilibrium phase, and such high energy would enable Arg292 to change its orientation. However, the inner orientation of Arg292 was maintained during the following simulation period, suggesting that this orientation was relatively stable in AspAT. The Arg292 side chain of AroAT formed a hydrogen bond with one water molecule and a salt bridge with the omega -carboxyl group of Glu141 in a side-by-side geometric fashion. Furthermore, this water molecule was fixed by the side chain of Asp15. We consider these interactions were the major forces that kept the Arg292 side chain at the edge of the substrate binding site.


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Fig. 8.   Structures of the active sites of simulated AspAT and AroAT. a, AspAT subunit A. b, AspAT subunit B. c, AroAT subunit A. d, AroAT subunit B. alpha -Aminoheptanoic acid is shown in orange, water molecules are shown as cyan spheres, and the others are colored by their atoms.

A difference distance matrix (48) has often been used to compare two structures. As superimposition of two structures is not required, this analytical technique is more likely to detect subtle differences than direct comparison of structural images. In order to compare the substrate binding sites of AspAT and AroAT, Delta rij and Delta Delta rij, which are the atomic displacement within one structure and difference between the displacements in two enzymes, respectively, were calculated. The Delta Delta rij values obtained were normalized by the average displacement values, and the matrix element was expressed by the following equations.
&Dgr;&Dgr;r<SUB>ij</SUB>=&Dgr;r<SUP><UP>Asp</UP></SUP><SUB>ij</SUB>−&Dgr;r<SUP><UP>Aro</UP></SUP><SUB>ij</SUB> (Eq. 10)
R<SUB>ij</SUB>=<FR><NU>&Dgr;&Dgr;r<SUB>ij</SUB></NU><DE><FENCE><FR><NU>&Dgr;r<SUP><UP>Asp</UP></SUP><SUB>ij</SUB>+&Dgr;r<SUP><UP>Aro</UP></SUP><SUB>ij</SUB></NU><DE>2</DE></FR></FENCE><SUP>2</SUP></DE></FR> (Eq. 11)
Delta rijAsp and Delta rijAro are the atomic displacements within the simulated AspAT and AroAT structures, respectively. The difference distance matrices calculated for subunits A and B are shown in the upper and lower triangles, respectively, of Fig. 9. A red spot indicates that the distance between two atoms was greater in AroAT than AspAT, and a blue one means it was shorter in AroAT than AspAT. There were many red and blue colonies in the matrix for subunit A, and they were thought to originate and derive from the different conformations of the Arg292 side chains of AspAT and AroAT. In subunit B, where the two side chains of Arg292 adopted similar conformations, substantial differences were detected between Asn142 and Arg292 as red colonies. The average distances between two residues in AspAT and AroAT were 6.2 and 9.5 Å, respectively. Detailed examination of the region around Asn142 revealed that the positions of the backbone structures had changed slightly and these changes may originate from the difference of residue 149, which is Ser in AspAT and Gly in AroAT.


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Fig. 9.   Difference distance matrices of atomic displacement in the active sites of simulated AspAT and AroAT. The matrix element (Rij) is calculated using the equation, Rij = (Delta ijAsp - Delta ijAro)/((Delta ijAsp + Delta ijAro)/2)2. The calculations for subunits A and B are shown in upper and lower triangles, respectively, and the color scale is shown at the bottom.

Van der Waals contact and dense packing of the substrate binding site are very important for hydrophobic substrate binding. Fig. 10 shows the numbers of all the atoms within 3-5 Å of the five carbon atoms of the sC7 side chain. No differences between the packing densities of AspAT and AroAT were detected. Both structures had about 13 non-hydrogen and 21 hydrogen atoms within the 5 Å layer around each carbon atom of their sC7 side chains, indicating that the densities of their interior were relatively high, as would be expected for typical globular proteins. These results suggest that both AspAT and AroAT had similar levels of van der Waals contact between the substrate and protein in their hydrophobic substrate-binding pockets and that the packing density was not a major determinant of the hydrophobic substrate specificities of these enzymes.


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Fig. 10.   Numbers of all the atoms within a given distance from each carbon atom of the alpha -aminoheptanoic acid side chain. a, AspAT; b, AroAT.

The mean square fluctuations about the positions of the Calpha atoms among the superimposed conformations were calculated on the basis of the averaged structures (Fig. 11). The averaged values over the whole subunit were 0.16 and 0.34 Å2 for AspAT and AroAT, respectively. Generally, the parts between the secondary structures showed higher than average values. Both large and small domains showed fluctuations of similar magnitudes, suggesting that both enzymes had adopted the closed conformation in the 100-ps time range. AroAT showed substantially greater fluctuations than AspAT, suggesting that AroAT is more flexible than AspAT, which has been suggested experimentally (3). Another interesting observation was that some of the dihedral angles of the sC7 side chain changed during simulation (each sC7 side chain has three dihedral angles). During a simulation of sC7 with water molecules, no such changes were observed within 1 ns.


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Fig. 11.   Mean square fluctuations of Calpha atoms. a, AspAT; b, AroAT. The helical and sheet regions are also shown. The coordinates from the trajectories during the last 100 ps were used for this analysis. Differences in position were calculated after every conformer during this 100-ps interval had been superimposed on the first structure.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Substrate Binding Energy-- It is possible to estimate the effect of one carbon in a substrate side chain by comparing the binding energies of substrates with different chain lengths. The energy contribution of one methylene group was about 1.4 kcal·mol-1, and the total contribution of the aliphatic side chain of sC7 was estimated to be 6 kcal·mol-1. From the difference between the affinity of a substrate and its cognate aliphatic acid inhibitor, the contribution of an amino group (Schiff base) was estimated to be 1 kcal·mol-1. The contribution of the alpha -carboxyl group of an amino acid substrate to binding was estimated by analyzing the Arg386 mutant, in which the residue recognizing the alpha -COO- group of the substrate was replaced by Lys, and found to be 1-2 kcal·mol-1 (49, 50). Therefore, the sum of the energy of sC7 is 8-9 kcal·mol-1, whereas the net binding energy of sC7 was only 3 kcal·mol-1. The binding energy is decreased by mixing free energy (2.3 kcal·mol-1). Furthermore, conformational change upon substrate binding costs energy 2-3 kcal·mol-1, estimated in studies on AspAT (51, 52). The total energy of substrate binding, 7.3-8.3 kcal·mol-1, is roughly consistent with the above sum.

Thermodynamic Cycle-- The solvent-transfer free energy is often used for analyzing hydrophobic interactions, and it accounts for some of the hydrophobic effect, i.e. the transfer of a substrate from water to a nonaqueous solvent. In terms of an enzyme-substrate interaction, not only the substrate, but also the enzyme, loses the unfavorable contact with the aqueous solvent in the pocket when the complex is formed. Therefore, the maximal effect is restricted to 2-fold, as much as can be derived from the solvent-transfer free energy in this model, which indicates that the hydrophobic binding pocket is also filled with aqueous solvent. In this study, the contributions of one methylene group to Delta Delta Gs, Delta Delta Hs, and TDelta Delta Ss in the forward reaction were calculated to be -1.4, -2.3, and -0.9 kcal·mol-1, respectively (Table IV). This Delta Delta Gs value is 1.5-fold the transfer free energy of one methylene group from water to pure hydrocarbon liquid (-0.9 kcal·mol-1) (53), suggesting that the interactions between AroAT and the aliphatic substrate side chains we studied were relatively strong. However, the thermodynamic parameters Delta Delta Gtrans, Delta Delta Htrans, and TDelta Delta Strans for the transfer of one methylene group have been estimated to be -1.0, -0.6, and 0.4 kcal·mol-1, respectively (53). From our thermodynamic cycle data, the thermodynamic parameters for hydrophobic substrate binding, Delta Delta GE-SBind, Delta Delta HE-SBind, and TDelta Delta SE-SBind, were calculated to be -0.4, -1.7, and -1.3 kcal·mol-1, respectively, when Delta Delta GEStrans was assumed to be zero (Scheme 3). The large negative Delta Delta HESBind value also suggests strong van der Waals interactions exist in the substrate-binding pocket of AroAT.


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Scheme 3.   Thermodynamic cycle. a, binding of substrate to enzyme. b, binding of one methylene group to enzyme. The dark shaded box indicates a protein, and the light shaded circle denotes a carbon of a hydrophobic substrate.

Morton et al. (10) performed a thorough analysis of hydrophobic ligand binding in the cavity of T4 lysozyme created by site-directed mutation. According to them, the entropic cost of one methylene group in the sC7 substrate for binding is -0.9 kcal·mol-1, if it is assumed that the substrate loses its translational, rotational and internal degrees of freedom (dihedral angle). The effect of the latter term of freedom must be viewed with caution, because Wynn et al. (54) found that the terminal carbon atoms in the flexible straight side chain of the structure (unnatural amino acid with hydrocarbon side chain) were disordered in their x-ray crystallographic study. However, this estimated value (-0.9 kcal·mol-1) may contribute to our observed value (-1.2 kcal·mol-1), the rest of which may be accounted for by fixations of the protein main and side chains. Such fixation (disorder-to-order transition) may be required for modulating the affinity and specificity of the enzyme (55).

The energetic contribution of one methylene group in the alkyl chain to the binding of AroAT is relatively large (-1.4 kcal·mol-1) and quite high in comparison with those reported in the literature: -0.4 kcal·mol-1 for T4 lysozyme (from the class III compounds in Ref. 10), -0.4 kcal·mol-1 for acyl-coenzyme A-binding protein (11), -0.8 kcal·mol-1 for Streptomyces griseus proteases (9, 56, 57), -1.0 kcal·mol-1 for Paracoccus denitrificans AroAT (58), and -1.2 kcal·mol-1 for alpha -chymotrypsin (56, 59). Even when compared with those for more closely related enzymes, the values were -0.73 and -1.2 kcal·mol-1 for AspAT and the chimeric enzyme constructed from AspAT and AroAT, respectively (3), it is quite high. Although more detailed comparisons are required for accuracy, AroAT may be one of the enzymes with very hydrophobic substrate-binding pockets. The above findings lead to the interpretation that the hydrophobic interactions are more efficient and/or the entropic costs of such interactions are lower with AroAT than other enzymes. Previous work has suggested that the energetic cost of conformational rearrangement of AroAT is relatively small, because this enzyme is more flexible than AspAT (3). Therefore, the latter interpretation seems feasible. Interesting phenomena have been reported with alpha -lytic protease and chymotrypsin; regions that are conformationally plastic and distinct from their substrate-binding sites play important roles in the substrate specificities of these enzymes (60-63). Recent studies by Rader and Agard (64) revealed that such plasticity is inherent and related to accommodating substrates, and they called it "dynamic close packing."

Averaged Structure of AroAT Complexed with sC7 Substrate-- In this study, the structures of AspAT and AroAT were re-built, using model building and MD simulation, as their bound forms with sC7 molecules. AroAT showed higher activities than AspAT toward not only aromatic substrates, but also aliphatic substrates. The activities of these two enzymes per hydrophobic unit of the substrate side chain differed about 2-fold. Therefore, their activities toward the sC7 substrate differed about 10,000-fold. Furthermore, it has been suggested that there is no striking steric hindrance, not even in the substrate binding site of AspAT, of sC7. For these reasons, we chose sC7 as the model substrate for the study to investigate the differences between the hydrophobic substrate specificities of AspAT and AroAT.

The orientation of the Arg292 side chain has been one of the main topics of the structural analysis carried out during the last two decades, because it is a most important and direct determinant of the recognition of dicarboxylic substrates (Table VI). In the presence of a dicarboxylic substrate, the guanidino group of Arg292 forms a salt bridge with the omega -carboxylic group of the dicarboxylate. When a substrate is not present, the side chain of Arg292 is generally oriented toward the outside of the substrate-binding pocket. Asp15 assists the orientations of Arg292 in both the open and closed forms of E. coli AspAT (29) and mitochondrial AspAT (mAspAT). However, cytosolic AspAT (cAspAT) has no aspartate residue in this position, it has been replaced by valine. Instead of Asp15, Glu141 interacts with Arg292 in the closed, but not the open, form of cAspAT (30). In the MD structure of AspAT, one of two Arg292 residues, which belong to each subunit, was directed toward the inside (Fig. 8a) and the other toward the outside of the substrate-binding pocket (Fig. 8b). Two conformations of Arg292 have been observed in the same subunit (occupancy is 0.5 for each conformer) of porcine cAspAT in the absence of a substrate analog (30). Arg292 has also been observed to direct its side chain toward the inner of the substrate-binding pocket of the PMP form of E. coli AspAT in the substrate-free state (46). These observations suggest that the outside-facing position of the Arg292 side chain in AspAT is not quite stable.

                              
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Table VI
Properties related to the orientation of the R292 side chain


The hexamutant of E. coli AspAT (V39L, K41Y, T47I, N69L, T109S, and N297S), which was constructed by Onuffer and Kirsch (65), is the nearest mutant to AroAT of those constructed so far. This hexamutant shows about 1,000 times higher activity than its parent AspAT toward aromatic substrates. Interestingly, the hexamutant adopts the closed conformation and its Arg292 side chain faces the inside, even in the absence a substrate (36), which indicates that the orientation of the Arg292 side chain is not always coupled to the open-closed conformational change. Seville et al. (34) proposed that, when a hydrophobic substrate is bound, Arg292 in AroAT is directed toward the outer side of the substrate-binding pocket in order to make room for large hydrophobic substrates, as has been observed in the complex structures formed between the AspAT hexamutant and aromatic substrate analogs. AroAT possesses both Asp15 and Glu141 residues, as discussed above, and there is interest in exactly how these residues interact with the Arg292 side chain in AroAT.

The MD structure of AroAT described above was relatively stable during 380-ps simulation at 300 K and the Arg292 side chain interacted with Asp15, Glu141 and a water molecule (some interactions are mediated by water molecules). These results suggest that the outward-facing position of the Arg292 side 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. The major difference is the presence of Glu141 in AroAT and Pro at the same position in AspAT. Previous research showed that Glu141 only has slight effect on the enzymatic activity of cAspAT (66). However, the importance of Glu141 in AroAT is still unclear, because Glu141 of AroAT can interact with Arg292 outside the substrate-binding pocket, whereas that of cAspAT may be not be able to do so. Therefore, if Glu141 really is important for the activity of AroAT, the positions of Arg292 and Glu141 in the substrate-free state will attract considerable interest.

Enzyme Flexibility-- In the enzyme-substrate complex, the hydrophobic interaction is entirely due to van der Waals interactions. Two mechanisms are thought to strengthen a hydrophobic interaction. One is to construct a substrate binding site from the hydrophobic residues, a notable example being retinal-binding protein (67), and the other is to increase the density of the substrate-binding pocket packing. In the cases of AspAT and AroAT, the former does not explain the different hydrophobic substrate specificities of the two enzymes, because their residues that interact directly with the substrate are almost fully conserved. The latter mechanism was explored using homology modeling and MD simulation. The results suggest that the substrate binding sites of both enzymes are densely packed and this is supported by the large enthalpy effects on substrate binding to AroAT we observed. Therefore, the latter mechanism is not directly responsible for hydrophobic substrate specificity either. However, an interesting observation was that the fluctuations of the main chain Calpha atoms of AroAT were larger than those of AspAT and large fluctuations would be expected to facilitate subtle rearrangements of the substrate-binding pocket (3). Therefore, the energetic cost of dense packing in AroAT may be lower than that in AspAT. Furthermore, large fluctuations of the entire AroAT molecule, or a large part of it, may enable rearrangement, not only in the vicinity of the pocket, but also of the majority of AroAT. Such global rearrangement may be a particularly effective strategy for reducing the steric hindrance and/or distortion of the molecular conformation induced by substrate binding.

    ACKNOWLEDGEMENT

We thank the Research Center for Protein Engineering, Institute for Protein Research, Osaka University, for performing the computer calculations.

    FOOTNOTES

* This work was supported in part by Grants-in-aid for Scientific Research 09680619 and 07558224 from the Ministry of Education, Science, Sports and Culture of Japan, and by a research grant from the Japan Society for the Promotion of Science ("Research for the Future" Program Grant JSPS-RFTF96L00506).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Research fellow of the Japan Society for the Promotion of Science.

§ To whom correspondence should be addressed. Tel.: 81-6-850-5433; Fax: 81-6-850-5442; E-mail: kuramitu{at}bio.sci.osaka-u.ac.jp.

1 The abbreviations used are: AroAT, aromatic amino acid aminotransferase; AspAT, aspartate aminotransferase; cAspAT, cytosolic AspAT; mAspAT, mitochondrial AspAT; sCn, alpha -aliphatic amino acid with a straight side chain bearing n carbon atoms; sCnI, aliphatic acid with a straight side chain bearing n carbon atoms; ksCn, aliphatic alpha -keto acid with a straight side chain bearing n carbon atoms; MD, molecular dynamic; PPY, N,5'-phosphopyridoxyl-L-tyrosine; PLP, pyridoxal 5'-phosphate; PMP, pyridoxamine 5'-phosphate.

2 The amino acid residues are numbered according to the sequence of pig cytosolic aspartate aminotransferase (68).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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