Ab Initio Molecular Orbital Calculations on N-β

J. Phys. Chem. B 1997, 101, 7001-7006

7001

Ab Initio Molecular Orbital Calculations on N-β-Mercaptoethylacetamide and Its Derivatives as Model Compounds of Coenzyme A (CoA), Acetyl-CoA, and Malonyl-CoA Young Kee Kang*,† and Seong Jun Han‡ Department of Chemistry, Chungbuk National UniVersity, Cheongju, Chungbuk 361-763, Korea ReceiVed: February 19, 1997; In Final Form: June 13, 1997X

The conformational study on N-β-mercaptoethylacetamide (MEA), acetyl-MEA, and malonyl-MEA as model compounds of coenzyme A (CoA), acetyl-CoA, and malonyl-CoA, respectively, is carried out using the ab initio method at the HF/6-31G* level and the hydration shell model in order to investigate the information on structures and energetics of the compounds when acetyl-CoA or malonyl-CoA is hydrolyzed into acetic acid and CoA or hydrogen malonate and CoA, respectively, in the gas phase and in solution. It is found that the overall preferred conformations of MEA are different from those of acetyl-MEA or malonyl-MEA and that the intramolecular hydrogen bond between amide hydrogen and terminal carboxylate oxygen is responsible for the low-energy conformations of malonyl-MEA. To determine the thermodynamic parameters in the gas phase at 298.15 K, the vibrational analysis was undertaken for minimized conformations. The preferred conformations and structural parameters of the three compounds are in accord with those of related compounds obtained from spectroscopic experiments and are reasonably consistent with the corresponding data of CoA and its derivatives complexed with CoA-binding proteins deduced from X-ray and NMR studies to within experimental uncertainty. For the hydrolysis of acetyl-MEA and malonyl-MEA in aqueous solution, total free energy changes (∆Gtot) are computed to be -12.4 and -22.5 kcal/mol, respectively, of which the former is in good agreement with the experimental value of -13.8 ( 0.1 kcal/mol for the hydrolysis of acetyl-CoA. Although computed free energy changes (ca. -15 kcal/mol) for the two reactions in the gas phase are almost the same, the hydration appears to raise and lower the ∆Gtot for the hydrolysis of acetyl-MEA and malonylMEA, respectively. The more negative value of ∆Gtot for the hydrolysis of malonyl-MEA in solution implies that the malonyl-CoA may have a higher group transfer potential than the acetyl-CoA.

Introduction Coenzyme A (CoA) is a prominent acyl carrier in a number of metabolic reactions and has a reactive thiol (-SH) group, to which acyl groups become covalently linked, forming thioesters.1a Because of relatively higher free energies of hydrolysis, thioesters have a high acyl group transfer potential, donating their acyl groups to a variety of acceptor molecules. Acyl derivatives of CoA are known to be involved in various essential pathways such as the citric acid cycle1a and fatty acid synthesis and oxidation.1b Acetyl-CoA is an important intermediate oxidized from glucose, fatty acids, and some amino acids in cellular respiration.1a Malonyl-CoA is known to be involved in biosynthesis of fatty acids.1b Recently, malonylCoA appears also to be a substrate for malonyl-CoA synthetase, which catalyzes the formation of malonyl-CoA directly from malonate and CoA in the presence of Mg2+ and ATP.2 However, nothing is completely known about the threedimensional structures of isolated CoA and its derivatives, except for an earlier 1H NMR investigation on CoA and acetyl-CoA,3,4 although several structures of CoA and its derivatives complexed with CoA-binding proteins have been determined by X-ray and NMR experiments.5 In particular, N-β-mercaptoethylacetamide (MEA) and acetyl-MEA have been used as model compounds for equilibrium thermodynamic experiments on hydrolysis of acetyl-CoA in aqueous solution.6 Several quantum mechanical calculations have been done for * To whom correspondence should be addressed. † Corresponding e-mail: [email protected]. FAX: +82431-273-8328. ‡ Present address: C&C Research Labs., 146-141 Annyung-ri, Taeanub, Hwasung-goon, Kyunggi-do 445-970, Korea. X Abstract published in AdVance ACS Abstracts, August 1, 1997.

S1089-5647(97)00618-4 CCC: $14.00

small thioesters in the gas phase to understand the hydrolysis of acetyl-CoA.7-9 Here we carried out the conformational study on MEA, acetylMEA, and malonyl-MEA as model compounds of CoA, acetylCoA, and malonyl-CoA, respectively, using the ab initio method and the hydration shell model in order to investigate the information on structures and energetics of the compounds when acetyl-CoA and malonyl-CoA are hydrolyzed in the gas phase and in solution. Methods The chemical structures and torsion angles of MEA, acetylMEA, and malonyl-MEA are shown in Figure 1. The initial bond lengths and bond angles adapted for the MEA moiety were taken from the structures of N-methylacetamide from an electron diffraction study10 and trans-ethanethiol from a microwave experiment.11 All ab initio calculations were carried out using the Gaussian 9212 and Gaussian 9413 molecular orbital packages run on the Cray YMP C90 supercomputer of the System Engineering Research Institute (SERI), Korea. Full geometry optimizations were performed at the direct HF/SCF level with the 3-21G and 6-31G* basis sets. These basis sets have been successfully used for conformational analysis of dipeptides.14-16 The 27 conformations of MEA generated by assigning three values of (60° and 180° to each torsion angle φ1, φ2, and φ3 were used as initial structures for full geometry optimization at the 3-21G level. The 10 3-21G optimized conformations were obtained, which were used as starting structures for MEA moieties of acetyl-MEA and malonyl-MEA. To a torsion angle φ4 of acetyl-MEA, three values of (60° and 180° were initially © 1997 American Chemical Society

7002 J. Phys. Chem. B, Vol. 101, No. 35, 1997

Kang and Han TABLE 1: Relative Energies, Thermal Energies, Entropies, Free Energies, and Torsion Angles of Minimum-Energy Conformations of N-β-Mercaptoethylacetamide (MEA) by HF/6-31G* Calculations at 298.15 Ka confb ggg g-g-g gtg gtgggt gg-gtggttt X-rayg 2ctsh 1eabi 1scuj

Figure 1. Chemical structures and torsion angles of (a) N-β-mercaptoethylacetamide (MEA), (b) acetyl-MEA, and (c) malonyl-MEA.

assigned. The three minimum-energy conformations of the malonyl sulfide anion optimized at the HF/6-31G** level17 were used as initial values of torsion angles φ4, φ5, and φ6 of malonylMEA. Thus, the 30 conformations were chosen as starting points for the HF/3-21G geometry optimization of acetyl-MEA and malonyl-MEA. The optimized structures at the HF/3-21G level were used as starting structures for the HF/6-31G* optimizations. In particular, only the eight low-energy structures of malonyl-MEA with a relative energy less than 10 kcal/mol were chosen as starting structures for further optimizations. Full geometry optimizations at all degrees of freedom were then carried out. To determine the thermodynamic parameters, vibrational frequencies were calculated for the fully optimized structures at the 6-31G* level with a relative energy less than 0.5 kcal/ mol. The thermal energy (ET), the entropy (S), and the enthalpy (H) were obtained from the frequencies and structures according to standard statistical mechanical treatments in the Gaussian 94 molecular orbital package at 298.15 K.18 Thus, the enthalpy change (∆H) and the Gibbs free energy change (∆Gg) in the gas phase at 298.15 K are given by

∆H ) ∆EHF + ∆ET + P∆V

(1)

∆Gg ) ∆H - T∆S

(2)

and

where ∆EHF is the HF energy change and P∆V is the work term equal to ∆nRT ) 0.592∆n kcal/mol. To estimate the hydration effects on the hydrolysis of acetylMEA and malonyl-MEA in aqueous solution, the hydration free energies (∆Ghyd) for optimized conformations were computed by using the hydration shell model of Kang et al.,19-22 which can reproduce well experimental hydration free energies of the neutral and charged organic compounds. The total free energy change (∆Gtot) in solution at 298.15 K is given by

∆Gtot ) ∆Gg + ∆∆Ghyd

(3)

∆EHFc

∆ETd

∆Se

∆Ggf

φ1

φ2

φ3

0.000 0.000 0.000 0.000 82.4 62.4 72.0 0.188 0.001 0.438 0.058 -81.1 -62.8 76.6 0.272 -0.009 0.664 0.065 82.1 178.8 70.3 0.838 80.7 179.6 -69.4 0.889 79.0 66.5 -169.4 1.721 104.8 -56.5 -51.1 2.321 141.4 68.6 -56.6 3.637 180.0 180.0 180.0 171.3 79.0 180.0 170.0 160.7 180.0 74.5 73.1 180.0

a All minimum-energy conformations are listed. Energies, entropies, free energies, and angles are in kcal/mol, cal/mol/K, kcal/mol, and degrees, respectively. Torsion angles are defined in Figure 1. Thermal energies and entropies were calculated only for conformations with ∆EHF e 0.5 kcal/mol. b Conformational states defined by torsion angles; see the text for details. c The HF/6-31G* zero of energy is -683.552 511 5 hartrees. d Thermal energy difference; ∆ET ) ET ET0, where ET0 ) Etrans0 + Erot0 + Evib0 ) 93.860 kcal/mol. e Entropy difference; ∆S ) S - S0, where S0 ) Strans0 + Srot0 + Svib0 ) 94.068 cal/mol/K. f Free energy difference in the gas phase; ∆Gg ) ∆EHF + ∆ET - T∆S. g Each X-ray conformation is denoted by a Brookhaven Protein Data Bank (PDB) entry code (ref 29). Data are complete as of July 1996. h X-ray structure of CoA complexed with citrate synthase (resolution 2.0 Å, ref 30). i X-ray structure of CoA complexed with dihydrolipoyl transacetylase (resolution 2.6 Å, ref 31). j X-ray structure of CoA complexed with succinyl-CoA synthetase (resolution 2.5 Å, ref 32); the second conformation of the compound is chosen because the two conformations are quite similar to each other.

Conformational states having minima near 60°, -60°, and 180° are denoted by the lower case letters g, g-, and t, respectively. The range of a torsion angle θ indicated by each letter code extends over a range of 120°; for example, g denotes 0° e θ < 120°, etc. In cases where the interval of torsion angle is -30° < θ < 30°, the conformational state is denoted by a letter c. Results and Discussion Minimum-Energy Conformations. a. MEA. From the 27 initial conformations of MEA, the 10 and 8 local minima are obtained at the 3-21G and 6-31G* levels, respectively. Minimumenergy conformations (MEC) of MEA at the 6-31G* level are listed in Table 1. Trans amide bonds are found to be favored in all MECs. The torsion angles φ1 and φ2 of the N-ethyl moiety are preferred in the gg and g-g- conformations, followed by gt. However, the energy of the gt conformation relative to the gg or g-g- conformation is less than 0.3 kcal/mol, which indicates that the ethyl linkage is flexible within limited regions. From 1H NMR experiments on CoA, Lee and Sarma reported that all three rotamers g, g-, and t for the -CH2-CH2- bond are equally populated,3,4 which is consistent with our results. In particular, the energies of trans conformations for the torsion angle φ1 are at least 2.3 kcal/mol higher than the gauche conformations. The torsion angle φ3 of the thiol group appears preferentially to be gauche and the trans conformation is about 0.9 kcal/mol higher in energy than the gauche conformation from a comparison of energies of the conformations ggg and ggt. These results are consistent with the results of microwave measurements on ethanethiol23,24 and 1-propanethiol.25 In particular, the conformations gg and g-g- for torsion angles φ2 and φ3 of the β-mercaptoethyl moiety are in good agreement with results

MO Calculations on N-β-Mercaptoethylacetamide

J. Phys. Chem. B, Vol. 101, No. 35, 1997 7003

TABLE 2: Relative Energies, Thermal Energies, Entropies, Free Energies, and Torsion Angles of Minimum-Energy Conformations of Acetyl-MEA by HF/6-31G* Calculations at 298.15 Ka confb

∆EHFc

∆ETd

∆Se

∆Ggf

φ1

φ2

φ3

φ4

gtg t tg-gt g-g-g-t ggg-t g-ggt tttt gtgc g-g-tc X-rayg 1acah 3mdei

0.000 0.061 0.068 0.991 1.600 3.532 4.957 5.769

0.000 0.009 -0.046

0.000 -3.309 -1.889

0.000 1.057 0.585

80.4 168.1 -77.7 78.4 -102.1 180.0 80.3 -75.6 117.9 -78.8

178.3 -67.4 -62.7 68.8 58.9 180.0 -178.2 -65.7 -92.4 -179.1

-79.3 91.3 -80.4 -96.8 70.1 180.0 86.6 172.6 81.5 160.2

-178.2 174.0 -176.2 -174.0 -167.2 179.9 11.3 -0.8 132.4 162.3

-

See footnotes a-g of Table 1. c The HF/6-31G* zero of energy is -835.328 716 1 hartrees. d ET0 ) 122.525 kcal/mol. e S0 ) 115.843 cal/ mol/K. h NMR structure of palmitoyl-CoA complexed with acyl-CoA-binding protein (ref 35). i X-ray structure of octanoyl-CoA complexed with acyl-CoA dehydrogenase (resolution 2.4 Å, ref 36). a-g

TABLE 3: Relative Energies, Thermal Energies, Entropies, Free Energies, and Torsion Angles of Minimum-Energy Conformations of Malonyl-MEA by HF/6-31G* Calculations at 298.15 Ka confb

∆EHFc

∆ETd

∆Se

∆Ggf

g g gtgc gggtgc tgtcgc ggg-cgc gtg-cgg tg-gcgg X-rayg 1buch

0.000 0.501 3.051 3.801 4.096 5.544

0.000 0.058

0.000 -0.749

0.000 0.782

- -

φ1

φ2

φ3

φ4

φ5

-95.7 80.3 -178.3 119.2 96.1 158.6 149.4 53.7

-65.9 43.6 72.2 84.3 155.5 -79.4 -144.4 141.8

88.1 56.4 -121.1 -115.4 -89.3 95.4 -91.5 90.3

-157.0 -155.7 -10.3 -5.1 -12.4 -24.1 -178.9 124.8

88.2 83.5 80.1 69.2 68.7 62.4 174.5 153.9

φ6 -2.8 2.4 -5.8 23.8 45.6 38.1

a-g See footnotes a-g of Table 1. c The HF/6-31G* zero of energy is -1022.400 045 7 hartrees. d ET0 ) 126.164 kcal/mol. e S0 ) 120.250 cal/mol/K. h Two X-ray structures of acetoacetyl-CoA complexed with butyryl-CoA dehydrogenase (resolution 2.5 Å, ref 37); the torsion angles φ6 are not shown because chemical structures of acetoacetyl and malonyl groups are different.

Figure 2. Lowest energy conformations of (a) MEA, (b) acetyl-MEA, and (c) malonyl-MEA obtained at the HF/6-31G* level. The hydrogen bond is represented by a broken line.

of the microwave investigation on 2-aminoethanethiol26 and the recent Raman experiment on 1-propanethiol.27 The first three MECs have comparable conformational energies, and the lowest energy conformation ggg is shown in Figure 2a. Although the thermal energies and entropies contribute somewhat to lower the relative conformational energies of the three MECs, the ordering of MECs does not changed (Table 1). No significant intramolecular electrostatic or hydrogenbond interactions appear to hold for these favored conformations, which is in accord with Raman and infrared investigations on MEA in solutions and in the solid phase.28 On the other hand, the first three MECs are not comparable to the X-ray structures tgt, ttt, and ggt of CoA complexed with its binding proteins29 such as citrate synthase,30 dihydrolipoyl transacetylase,31 and succinyl-CoA synthetase,32 respectively. Although the conformations ggt and ttt appear to have relative conformational energies of 0.9 and 3.6 kcal/mol, respectively, at the 6-31G* level, the discrepancy in the computed conformations and X-ray structures may arise from one or both of the following causes: (1) the long-range intra- and intermolecular interactions which the isolated MEA cannot have and (2) different environments in the gas phase and in the crystalline state.

b. Acetyl-MEA. The nine and eight local minima are obtained at the 3-21G and 6-31G* levels, respectively, starting from 30 initial conformations. In Table 2 MECs of acetyl-MEA obtained at the 6-31G* level are listed. All amide bonds are trans, similar to those of MEA. The preferred conformations for the torsion angles φ1 and φ2 of the N-ethyl group are gt, tg-, and g-g-, of which the first two conformations are not preferred in MEA. In particular, the preference of the trans conformation for a torsion angle φ1 or φ2 of acetyl-MEA is remarkable, because its energy is relatively higher in MEA (see Tables 1 and 2). This implies that the acetylation of MEA yields a change in the population of conformations of the -CH2-CH2- bond that are equally populated in MEA, which is consistent with the results of 1H NMR experiments on CoA and acetyl-CoA.4 The torsion angle φ3 of the ethyl sulfide linkage is preferred in the conformations g- and g. It appears that the relative stability of the g- conformations is increased and the trans conformation is highly forbidden by introducing the acetyl group (see Tables 1 and 2). This is similar to the results of ab initio calculations on ethyl thioformate with the 3-21G* basis set.8 The conformation for the S-COCH3 bond (i.e., the torsion angle

7004 J. Phys. Chem. B, Vol. 101, No. 35, 1997

Kang and Han

TABLE 4: Structure Parameters of Lowest Energy Conformations of MEA, Acetyl-MEA, and Malonyl-MEA Obtained at the HF/6-31G* Level MEA parametera

this work

C1-C2 C2-O1 C2-N N-C3 C3-C4 C4-S S-C5 C5-O2 C5-C6 C6-C7 C7-O3 C7-O4 C1-H1 C1-H2 C1-H3 N-H4 C3-H5 C3-H6 C4-H7 C4-H8 S-H9 C6-H9 C6-H10 C6-H11

1.513 1.201 1.357 1.444 1.528 1.825

C1-C2-O1 C1-C2- N O1-C2-N C2-N-C3 N-C3-C4 C3-C4-S C4-S-C5 S-C5-C6 S-C5-O2 C5-C6-C7 C6-C7-O3 C6-C7-O4 C2-N-H4 C2-C1-H1 C2-C1-H2 C2-C1-H3 N-C3-H5 N-C3-H6 C3-C4-H7 C3-C4-H8 C4-S-H9 C5-C6-H9 C5-C6-H10 C5-C6-H11

122.2 115.3 122.5 121.6 113.9 114.4

1.080 1.084 1.086 0.994 1.081 1.084 1.083 1.082 1.328

exptlb

acetyl-MEA this work

Bond Length, Å 1.532 1.514 1.242 1.202 1.329 1.353 1.449 1.448 1.516 1.528 1.804 1.816 1.783 1.189 1.511

1.010 1.090 1.090 1.090 1.090 1.340

exptlc 1.528 1.226 1.345 1.468 1.525 1.813 1.798 1.220 1.519

1.081 1.084 1.085 0.993 1.080 1.080 1.080 1.081 1.081 1.084 1.085

118.3 108.9 112.3 109.1 107.8 108.3 110.3 109.6 97.8

Bond Angle, deg 120.0 121.9 120.1 116.9 115.8 113.2 123.1 122.3 126.8 122.6 122.3 119.6 111.8 111.7 105.8 112.2 113.0 113.9 100.9 103.6 114.1 117.7 122.6 120.6

118.7

107.9 109.5 109.7 107.4 109.5

119.3 108.8 113.0 108.7 107.9 108.8 110.8 109.9 108.7 110.7 109.8

malonyl-MEA this work 1.514 1.211 1.345 1.440 1.532 1.819 1.808 1.187 1.500 1.573 1.218 1.240 1.084 1.081 1.086 1.007 1.085 1.081 1.080 1.085

exptld 1.512 1.234 1.393 1.452 1.521 1.823 1.768 1.229 1.514 1.519 1.230

1.083 1.085

121.2 115.1 123.7 121.6 113.4 112.8 101.5 113.9 121.4 111.9 113.9 115.9 118.0 108.1 112.8 108.4 107.9 108.9 110.8 109.9

120.7 117.6 121.9 120.7 112.8 113.1 106.3 108.9 113.8 114.5 120.1

108.4 111.3

a Refer to Figure 1 for atom labeling. b Average of three X-ray structures of CoA complexed with proteins, taken from refs 30-32; see footnotes h-j of Table 1. c Average of NMR and X-ray structures of palmitoyl-CoA and octanoyl-CoA complexed with proteins, respectively, taken from refs 35 and 36; see footnotes h and i of Table 2. No coordinates of H atoms are reported from the X-ray study. d Average of two X-ray structures of acetoacetyl-CoA complexed with the protein, taken from ref 37; see footnote h of Table 3. No coordinates of H atoms are reported from the X-ray study.

φ4) exists preferentially in trans, being ca. 5 kcal/mol more stable than the cis conformation (i.e., the E conformation), which is in accord with the recent ab initio calculations on methyl thioacetate at the 6-31++G** and MP2/6-31++G** levels9 and with the results for the O-COCH3 bond of ethyl acetate obtained from ultrasonic relaxation33 and IR34 experiments. The first three MECs have comparable relative energies less than 1 kcal/mol, which are different from the preferred con-

TABLE 5: Energies, Thermal Energies, Entropies, and Hydration Free Energies of Compounds at the HF/6-31G* Level Used in Calculating Free Energies of Hydrolysis at 298.15 Ka compound

EHFb

ETc

Sd

∆Ghyde

MEA acetyl-MEA malonyl-MEA hydrogen malonate acetic acid H2O

-683.552 253 3 -835.328 689 0 -1022.399 836 9 -414.877 879 7 -227.810 647 8 -76.010 746 5

93.9 122.5 126.2 48.0 44.7 16.2

94.5 115.3 120.1 79.9 67.6 46.4

-5.8 -8.8 -76.9 -84.7 -6.7f -6.3g

a For MEA, acetyl-MEA, and malonyl-MEA, each of four thermodynamic quantities in the table was calculated by statistically averaging over the conformations with the ∆EHF e 0.5 kcal/mol. See the text for details. For hydrogen malonate, acetic acid, and H2O, these quantities were calculated for the lowest energy conformations, whose starting conformations were taken from the results of ref 17 obtained at the HF/6-31G** level. b HF energies in hartrees; 1 hartree ) 627.509 551 6 kcal/mol. c Thermal energies in kcal/mol; ET ) Etrans + Erot + Evib. d Entropies in cal/mol/K; S ) S e trans + Srot + Svib. Hydration free energies in kcal/mol, calculated by using the hydration shell model of Kang et al. (refs 19-22) for low-energy conformations optimized at the HF/6-31G* level. For acetic acid and H2O, experimental values of hydration free energy were used (see the text). f Taken from ref 22. g Taken from ref 40.

formations of MEA, and the lowest energy conformation gtg-t of acetyl-MEA is shown in Figure 2b. The contribution of the thermal energies and entropies of acetyl-MEA to its conformational free energies appears to be more significant than MEA, but the lowest energy conformation is still the lowest free energy conformation. As seen in MEA, no significant intramolecular electrostatic or hydrogen-bond interactions appear to hold for the favored conformations of acetyl-MEA. The third lowest free energy conformation tg-gt is comparable to the structure of palmitoyl-CoA complexed with acyl-CoA-binding protein deduced from NMR experiments,35 but no MEC of acetyl-MEA is matched with the X-ray structure of octanoylCoA complexed with acyl-CoA dehydrogenase.36 This agreement and discrepancy may be explained by the same causes that are discussed for MEA. c. Malonyl-MEA. From the 27 starting conformations of malonyl-MEA, the 16 and 6 local minima are obtained at the 3-21G and 6-31G* levels, respectively. MECs of malonyl-MEA at the 6-31G* level are listed in Table 3. Only the first two MECs have relative energies less than 3 kcal/mol and seem to be feasible. All amide bonds are trans, similar to those of MEA and acetyl-MEA. The torsion angles φ1, φ2, and φ3 of the first two MECs of malonyl-MEA are similar to those of the second and first MECs of MEA, respectively. This implies that the preferred conformations of the S-ethylacetamide moiety of malonyl-MEA are similar to those of MEA. On the other hand, MECs of this moiety of acetyl-MEA are no longer to be preferred in malonylMEA. However, the torsion angles φ3 and φ4 of the sulfide linkage of malonyl-MEA are preferentially gt, which is similar to those of acetyl-MEA. These calculated results indicate that the overall preferred conformations of malonyl-MEA are different from those of MEA or acetyl-MEA. Different from that found in the cases of MEA and acetylMEA, the intramolecular hydrogen bond between the carboxylate oxygen and the amide hydrogen seems to play a major role in determining the first two MECs of malonyl-MEA, whose distances are 1.93 and 1.88 Å for the first and second conformations, respectively. The corresponding angles ∠NH4‚‚‚O4 are 171° and 170°. This implies that this hydrogen bond appears to lead the most preferred conformation of malonyl-MEA to be different from that of MEA or acetyl-MEA.

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J. Phys. Chem. B, Vol. 101, No. 35, 1997 7005

TABLE 6: Changes in Energies, Enthalpies, Entropies, Free Energies, Hydration Free Energies, and Total Free Energies for Hydrolysis of Acetyl-MEA and Malonyl-MEA at 298.15 Ka reactantb

∆EHFc

∆ETd

∆He

∆Sf

∆Ggg

∆∆Ghydh

∆Gtoti

acetyl-MEA malonyl-MEA

-14.7 -12.3

-0.2 -0.5

-14.9 -12.8

0.4 8.0

-15.0 -15.2

2.6 -7.3

-12.4 (-13.8 ( 0.1)j -22.5

a Energies and free energies in kcal/mol; entropies in cal/mol/K. b The first-row reaction, acetyl-MEA + H2O f MEA + acetic acid; the secondrow reaction, malonyl-MEA + H2O f MEA + hydrogen malonate. c,d,f,h Changes in HF energies, thermal energies, entropies, and hydration free energies, respectively, which were calculated by using values in Table 5. e Enthalpy changes given by eq 1 of the text. g Free energy changes in the gas phase given by eq 2 of the text. i Total free energy changes in aqueous solution given by eq 3 of the text. j Experimental value estimated by using the free energy of hydrolysis of acetyl-CoA at pH 7.0 (ref 6) and the dissociation constant of acetic acid at 25 °C (ref 41).

In addition, the loss of preferred conformations for the torsion angle φ5 of the malonyl methyl sulfide anion, studied by us previously,17 supports the important role of this hydrogen bond in determining the preferred conformations of malonyl-MEA. The contribution of the thermal energies and entropies of malonyl-MEA to its conformational free energies seems to be less effective than acetyl-MEA and comparable to that of MEA. The lowest energy conformation g-g-gtgc is conserved as the lowest free energy conformation, which is shown in Figure 2c. No MEC obtained at the 6-31G* level is consistent with X-ray structures of acetoacetyl-CoA complexed with butyryl-CoA dehydrogenase.37 The same causes may be applied to this disagreement as seen in MEA and acetyl-MEA. Structural Parameters. In Table 4 we list the structural parameters of the lowest energy conformations of MEA, acetylMEA, and malonyl-MEA optimized at the 6-31G* level. The corresponding experimental values are also shown in Table 4, which were obtained by averaging bond lengths and bond angles of CoA and its derivatives complexed with CoA- or acyl-CoAbinding proteins deduced from X-ray30-32,36,37 and NMR35 experiments. The optimized bond lengths and bond angles of the three compounds agree with each other to within 0.01 Å and 1°, respectively, except for the S-C5 and N-H4 bond lengths and the O1-C2-N, N-C3-C4, and C2-N-H4 bond angles of acetyl-MEA and malonyl-MEA that may be due to the intramolecular hydrogen bond in malonyl-MEA. In addition, the 6-31G* optimized bond lengths and bond angles of three compounds are, in general, consistent with averaged experimental values to within 0.03 Å and 3°, respectively. Exceptions are as follows: (1) the differences in bond lengths are 0.04 Å for the C2-O1 bond of MEA and 0.040.05 Å for the bonds C2-N, S-C5, C5-O2, and C6-C7 of malonyl-MEA, and (2) the differences in bond angles are 4-6° for the bond angles O1-C2-N, N-C3-C4, and S-C5-C6 of acetyl-MEA and 5-8° for the bond angles C4-S-C5, S-C5C6, S-C5-O2, and C6-C7-O3 of malonyl-MEA. In particular, the C4-S-H9 bond angle of MEA is computed to be 97.8°, while the corresponding X-ray value is 109.5°, and this ab initio value is, however, consistent with the values determined from microwave experiments on ethanethiol,11,23,24 1-propanethiol,25 and 2-aminoethanethiol.26,38 Of course, the disagreement between the calculated and X-ray geometries can be drawn by the causes discussed above, but the differences in geometries are less than the uncertainty in the observed data if it is taken into account that crystallographic R factors of X-ray structures of CoA complexes considered here are 2.0-2.6 Å.30-32,36,37 In addition, the computed geometries of the -CCH2SH moiety of MEA are in accord with those of ethanethiol,11,23,24 1-propanethiol,25 and 2-aminoethanethiol26,38 determined from microwave investigations. The 6-31G* optimized geometries of the -CSCOCH3 moiety of acetyl-MEA are in good agreement with those of methyl thioformate determined from infrared and microwave experiments39 and those of methyl thioacetate and ethyl thiopropanoate optimized

at the HF/6-31++G* level.9 The optimized geometries of the malonyl sulfide moiety at the 6-31G* level are also in accord with those of the malonyl sulfide anion optimized recently by us at the HF/6-31G** and HF/6-31+G** levels.17 This agreement between the geometries computed at the 6-31G* level and those of previous spectroscopic measurements and ab initio calculations supports the validity of the results reported here. Hydrolysis of Acetyl-MEA and Malonyl-MEA. As described in the Introduction, thioesters have a high acyl group transfer potential because of higher free energies of hydrolysis.1a The free energy change involved in the hydrolysis of acyl-CoA is a measure of the amount of free energy stored in the thioester bond. The hydrolysis of acetyl-MEA and malonyl-MEA can be expressed by acetyl-MEA + H2O f MEA + acetic acid (1) and malonyl-MEA + H2O f MEA + hydrogen malonate (2), which may be models of the hydrolysis of acetyl-CoA and malonyl-CoA, respectively. The necessary quantities to calculate the free energy changes of hydrolysis are listed in Table 5, which correspond to statistically weighted values for low-energy conformations of MEA, acetyl-MEA, and malonyl-MEA at the 6-31G* level, as described later. The values of hydrogen malonate, acetic acid, and H2O were calculated for their lowest energy conformations optimized at the same basis set, which were started from lowest energy conformations optimized at the HF/6-31G** level.17 In addition, the hydration free energies of the compounds were computed using the hydration shell model of Kang et al.19-22 in order to figure out solvation effects on the hydrolysis. However, because the hydration shell model underestimates the hydration free energy of acetic acid and cannot be applied to H2O,22 experimental values were used for acetic acid and H2O, taken from refs 22 and 40, respectively. Because the first few low-energy conformations for MEA, acetyl-MEA, and malonylMEA have comparable energies as seen in Tables 1-3, four thermodynamic quantities in Table 5 were statistically averaged over conformations with ∆EHF e 0.5 kcal/mol for each compound. The total free energies in the hydrated state, given by eq 3, were used to compute normalized statistical weights. In Table 6 the changes in HF energies, thermal energies, enthalpies, entropies, free energies in the gas phase, hydration free energies, and total free energies in aqueous solution for the hydrolysis of acetyl-MEA and malonyl-MEA at 298.15 K are listed. The changes in enthalpies and free energies in the gas phase and in solution were calculated according to eqs 1-3 of the text. The gas-phase free energy changes for the hydrolysis of acetyl-MEA and malonyl-MEA are computed to be -15.0 and -15.2 kcal/mol, respectively, for the reactions 1 and 2 described above. Although there is a difference of 2.4 kcal/mol in HF energies, the entropic term compensates it and the final free energy changes become almost the same. The corresponding free energy changes in solution are computed to be -12.4 and -22.5 kcal/mol, respectively, of which the hydration contributes to raise and lower the ∆Gtot of acetylMEA and malonyl-MEA, respectively. The value for the hydrolysis of acetyl-MEA is in good agreement with the

7006 J. Phys. Chem. B, Vol. 101, No. 35, 1997 experimental value of -13.8 ( 0.1 kcal/mol for the hydrolysis of acetyl-CoA, which was estimated by using the free energy of hydrolysis of acetyl-CoA at pH 7.06 and the dissociation constant of acetic acid at 25 °C.41 The more negative value of ∆Gtot for the hydrolysis of malonyl-MEA in solution implies that the malonyl-CoA may have a higher group transfer potential than the acetyl-CoA. Conclusions From the conformational study on MEA, acetyl-MEA, and malonyl-MEA as model compounds of CoA, acetyl-CoA, and malonyl-CoA, respectively, carried out using the ab initio method at the HF/6-31G* level and the hydration shell model, it is found that the overall preferred conformations of MEA are different from those of acetyl-MEA or malonyl-MEA and that the intramolecular hydrogen bond between amide hydrogen and terminal carboxylate oxygen is responsible for the low-energy conformations of malonyl-MEA. Although the contributions from thermal energies and entropic terms to free energies are not negligible, the lowest energy conformations of the three compounds are conserved as the lowest free energy conformations in the gas phase at 298.15 K. The preferred conformations and structural parameters of the three compounds are in accord with those of related compounds obtained from spectroscopic experiments and are reasonably consistent with the corresponding data of CoA and its derivatives complexed with CoA-binding proteins deduced from X-ray and NMR studies to within experimental uncertainty. For the hydrolysis of acetyl-MEA and malonyl-MEA in aqueous solution at 298.15 K, total free energy changes (∆Gtot) are computed to be -12.4 and -22.5 kcal/mol, respectively, of which the former is in good agreement with the experimental value of -13.8 ( 0.1 kcal/mol for the hydrolysis of acetylCoA. Although computed free energy changes (ca. -15 kcal/ mol) for the two reactions in the gas phase are almost the same, the hydration appears to raise and lower the ∆Gtot for the hydrolysis of acetyl-MEA and malonyl-MEA, respectively. The more negative value of ∆Gtot for the hydrolysis of malonylMEA in solution implies that the malonyl-CoA may have a higher group transfer potential than the acetyl-CoA. Acknowledgment. This work was partially supported by the Science and Technology Policy Institute, Korea (1996) and the Korea Science and Engineering Foundation (Grant No. 92-2400-06). The authors thank the System Engineering Research Institute (SERI), Korea, for use of the Cray YMP C90 supercomputer. References and Notes (1) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed.; Worth Publishers: New York, 1993; (a) Chapter 15; (b) Chapter 20. (2) Kim, Y. S.; Chae, H. Z. Biochem. J. 1991, 273, 511. (3) Lee, C.-H.; Sarma, R. H. FEBS Lett. 1974, 43, 271. (4) Lee, C.-H.; Sarma, R. H. In Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions; Sundaralingam, M., Rao, S. T., Eds.; University Park Press: Baltimore, 1975; p 631. (5) Engel, C.; Wierenga, R. Curr. Opin. Struct. Biol. 1996, 6, 790. (6) Jencks, W. P.; Gilchrist, M. J. Am. Chem. Soc. 1964, 86, 4651. (7) Hayes, D. M.; Kenyon, G. L.; Kollman, P. A. J. Am. Chem. Soc. 1978, 100, 4331.

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