158
Biochimica et Biophysica Acta, 1036 (1990) 158-161 Elsevier
BBA
BBAGEN 20269
Report
Histamine tautomerism and its mode of action Graham A. Worth, Paul M. King and W. Graham Richards Oxford Centre for Molecular Sciences and Physical Chemistry Laboratory, Oxford ( U.K.) (Received 19 March 1990)
Key words: Histamine; Receptor, Ha; Molecular orbital calculation; Free energy perturbation
An established combination of quantum mechanical calculations and molecular dynamics simulations (Worth, C.A., King, P.M. and Richards, W.G. (1989) Biochim. Biophys. Acta 993, 134-136; Cieplak, P., Singh, U.C. and Kollman, P.A. (1987) Int. J. Quant. Chem. QBSI4, 65-74; Reynolds, C.A., King, P.M. and Richards, W.G. (1988) Nature 334, 80-82) has been used to calculate the tautomer ratios of histamine species in aqueous solution. The results are in good agreement with experiment and provide a bridge between experimental data and earlier theoretical calculations. Despite its importance, the molecular mechanism of histamine action is not well understood. Blockade of its effect at histamine H 2 receptors accounts for several of the world's leading drugs, yet details of the receptor site and mechanism remain elusive. The experimental work of Black et al. [4] and the theoretical gas phase calculations of Weinstein et al. [5,6] have, however, implicated tautomerism in the H a receptor site mechanism. Histamine has two possible tautomers, the proximal (or) and distal (~') forms. Although other species are present in solution, the proposed mechanism involves tautomerism of the amino protonated monocation and free base forms. HC
C/'R
I
(
'
HC
I
C/R
I
N%c/NH
I
HN~c~N
H
H
N(w)H
N(~)H
R = CH2CHzNH 2
Free Base
+
R = CH2CH2NH 3
Monocation
The ratio of tautomers can be described by the equilibrium constant K T = [N(~')H]/[N(~r)H]
(1)
or the corresponding difference in Gibbs free energy, AG T.
Abbreviation: FEP, free energy perturbation. Correspondence: W.G. Richards, Physical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, U.K.
Using the energy cycle below, the calculation of AGT(aq) has two parts to it. First the difference in energy between the tautomers in the gas phase is calculated. The tautomers are then solvated and the difference in free energy of hydration between them is computed using the free energy perturbation (FEP) method [7,81. AGT(g)
N(rr)H (g)
,
N('r)H (g)
AG1 (aq)
N(~')H (aq)
~ N(,r)H (aq)
To calculate AGT(g ), molecular enthalpies at 0 K were calculated using the G A U S S I A N 86 [9] and C A D P A C [10] ab initio programs with various basis sets, and then corrected to Gibbs energies at 298 K using thermodynamic data from A M P A C [11]. Geometries for the ab initio studies were fully optimised up to the 6-31 G level, starting from the known crystal structure of both the histamine base [12] and the monocation [13]. The difference in Gibbs free energy of hydration of the two tautomers was calculated using the FEP module of the A M B E R [14] suite of programs, mutating the Tr tautomer into the z tautomer in solution during the course of a molecular dynamics simulation, and calculating the free energy change. The molecular mechanics parameters for the histamine tautomers were taken from the A M B E R all atom force field [15]. Charges were derived by fitting to the molecular electrostatic potential obtained from the 631G ab initio wavefunction using the program Q U E S T [14]. The starting tautomer, in a box of TIP3P water molecules [16] was first minimized and then equi-
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
159 Table I
The oariation a.u. = 2625.35 in k J . mol - 1, basis sets and
in tautomer ab initio gas phase enthalpies, in a.u., (1 k J . m o l - 1) and the difference in tautomer enthalpy, A H r at 0 K for histamine monocation and base using different geometries
Basis set
A b initio energies in a.u.
A H . r ( g ' 0) ( k J m o l - 1)
N( ~')H
N ( or ) H
a) H i s t a m i n e b a s e RHF/STO-3G//a
- 353.44832
- 353.44968
3.56
RHF/3-21G//b
- 355.90289
- 355.90249
- 1.05
RHF/6-31G//c RHF/6-31G *//c
- 357.74957 - 357.89988
- 357.74828 - 357.89920
- 3.40 - 1.79
RHF/6-31G * *//c
- 357.92232
- 357.92158
- 1.94
MP2/6-31G *//c MP2/6-31G * *//c
- 359.02716 - 359.10541
- 359.02623 - 359.10447
- 2.45 - 2.48
RHF/STO-3G//a
- 353.90906
- 353.89247
- 42.55
RHF/3-21G//b
- 356.29941
- 356.27894
- 53.76
RHF/6-31G//c
- 358.13425
- 358.11319
- 55.30
RHF/6-31G *//c
- 358.28819
- 358.26993
- 47.94
RHF/6-31G * *//c
- 358.31282
- 358.29440
- 48.38
MP2/6-31G *//c MP2/6-31G * *//c
- 359.41381 - 359.49639
- 359.39455 - 359.47718
- 50.56 - 50.42
b) H i s t a m i n e m o n o c a t i o n
Basis set [22-26] n o t a t i o n is e n e r g y calculation m e t h o d / / g e o m e t r y calculation m e t h o d . M o l e c u l a r g e o m e t r y was f o u n d f r o m e n e r g y minim i z a t i o n calculations using a restricted H a r t r e e - F o c k ( R H F ) m e t h o d a n d (a) S T O - 3 G , (b) 3 - 2 1 G or (c) 6-31G. M P 2 shows that the c a l c u l a t i o n was p e r f o r m e d using Moller-Plesset 2nd o r d e r p e r t u r b a tion theory.
librated by molecular dynamics simulation for 4.5 ps. The dynamics were performed using periodic boundary conditions at constant temperature and pressure, using SHAKE [17] to constrain all bonds and a non-bonded cutoff of 0.8 nm (histamine base) or 0.9 nm (histamine monocation). The perturbation was carried out over 21 separate simulations (or windows) with a mutation parameter of AX = 0.05. At each window, 500 steps (1 ps) of equilibration were followed by 2000 steps (4 ps) of data collection. The methods used are reported in more detail in an earlier paper which used the same techniques to calculate the tautomer ratio of 4(5)-methylimidazole in aqueous solution [1]. The results of the ab initio gas phase calculations are shown in Table I. All the basis sets used, with one exception, predict that both the histamine base and the histamine monocation are more stable in the N(~')H tautomer, with the preference being far greater for the monocation. In contrast, the STO-3G result, in agreement with Topiol et al [18], predicts that histamine base is more stable in the N(~r)H form. Table II shows the FEP results, which are the difference in free energy of hydration at 298 K between the tautomers, AAG(hyd) = AG(hyd, 'r) - AG(hyd, rr)
(2)
Taking into account the FEP errors, there is very little difference in the free energies of hydration of the histamine base tautomers, although the N(~')H appears to be slightly more strongly solvated. In contrast hydration favours the N(~r)H histamine monocation over the N(~')H form. Presumably protonation of the side-chain changes the differences in solvation energies due to changing the accessibility of hydrogen bonding sites to the aqueous solvent. The final results (Table III) show the difference in Gibbs free energy between the tautomers in both the gas and solution phases at 298 K. Experimentally determined results are also given for comparison. It can be seen that with good quality basis sets the calculated ratio of histamine base tautomers is in good agreement with the only experimental figure available. The histamine monocation calculation is not in such good agreement with experiment, but it is qualitatively correct: the calculated difference in hydration energy decreases the gas phase energy difference to give a AG v much closer to experiment. Given the good agreement of the histamine base and earlier 4(5)-methylimidazole results [1] seems likely that the most significant error is the underestimation of the coulombic interactions for the monocation by implementing a non-bonded cutoff in the simulations. It may also be significant that the only experiment which measured the ratio in histamine itself, rather than that of the N-methyl derivatives, was that of Wasylishen and Tomlinson [19], who found the ratio to be much more in favour of the N(~-)H tautomer than previous experiments, but were unable to give an exact value. Table II
The F E P results T w o i n d e p e n d e n t F E P r u n s w e r e m a d e on each system. R u n 1 is for the p e r t u r b a t i o n N(~r)H---, N O - ) H ; r u n 2 is for NO-)H---, N ( ~ r ) H . Both these i n d e p e n d e n t p e r t u r b a t i o n s w e r e r u n twice, in the f o r w a r d a n d then the reverse directions. E a c h p e r t u r b a t i o n also c a l c u l a t e s the e n e r g y in b o t h directions. This n u m b e r of s i m u l a t i o n s allows the statistical errors i n v o l v e d in the calculations to be a s c e r t a i n e d * Figures are A A G ( h y d ) in k J . m o 1 - 1 N ( ~ r ) H --} N ( z ) H . Run 1
and
given for the m u t a t i o n
Run 2
a) H i s t a m i n e base Forward - 2.04 Backward - 3.81 Average - 2.93 A A G ( h y d ) = - 1.79 + 1.36 k J-
__.0.67 + 0.35 + 0.76 mol - 1
b) H i s t a m i n e m o n o c a t i o n Forward 32.51 +__0.57 Backward 29.66 + 0.31 Average 31.09 + 0.65 A A G ( h y d ) = 32.71 + 2 . 2 6 k J - m o 1 - 1
- 1.78 + 1.10 0.49 + 0.25 - 0.65 + 1.13
35.76 -t- 0.90 32.88 + 1.97 34.32 + 2.17
* E r r o r in each r u n is the s t a n d a r d d e v i a t i o n b e t w e e n the two energies c a l c u l a t e d d u r i n g the p e r t u r b a t i o n . A v e r a g e errors o v e r the i n d e p e n d e n t runs a n d all r u n s are also s h o w n .
160 Table I1t
Difference in free energies, in kJ. tool 1, for the tautomeric shift N(~r)H --* N('r)H for histamine base and histamine monocation The accuracy of the AG(aq, 298) results are, given the inherent errors in the gas phase calculations, dependant on the statistical errors in the FEP results given in Table 1I. Basis set
Histamine base
Histamine monocation
AG(g, 298) AG(aq, 298) AG(g, 298) AG(aq, 298) (a) Calculated * AM127 RHF/STO-3G//a RHF/3-21G//b RHF/6-31G//c RHF/6-31G*//c RHF/6-31G * * / / c MP2/6-31G * / / c MP2/6-31G * * / / c (b) Experimental * * Charton [28] Ganellin [29] Reynolds [20] Waylishen [19]
**
- 2.17 3.77 - 0.83 -3.18 - 1.57 - 1.71 -2.23 - 2.25
- 3.95 2.00 - 2.61 -4.96 - 3.35 - 3.50 -4.00 - 4.03
--- - 3.4
34.56 - 42.15 - 52.35 -53.89 - 46.53 - 46.97 -49.15 49.02
67.28 - 9.43 - 19.64 -21.18 - 13.82 - 14.26 -16.44 - 16.30
- 2.1 - 3.7 = - 3.4
Biochimica et Biophysica Acta, 1036 (1990) 158-161 Elsevier
BBA
BBAGEN 20269
Report
Histamine tautomerism and its mode of action Graham A. Worth, Paul M. King and W. Graham Richards Oxford Centre for Molecular Sciences and Physical Chemistry Laboratory, Oxford ( U.K.) (Received 19 March 1990)
Key words: Histamine; Receptor, Ha; Molecular orbital calculation; Free energy perturbation
An established combination of quantum mechanical calculations and molecular dynamics simulations (Worth, C.A., King, P.M. and Richards, W.G. (1989) Biochim. Biophys. Acta 993, 134-136; Cieplak, P., Singh, U.C. and Kollman, P.A. (1987) Int. J. Quant. Chem. QBSI4, 65-74; Reynolds, C.A., King, P.M. and Richards, W.G. (1988) Nature 334, 80-82) has been used to calculate the tautomer ratios of histamine species in aqueous solution. The results are in good agreement with experiment and provide a bridge between experimental data and earlier theoretical calculations. Despite its importance, the molecular mechanism of histamine action is not well understood. Blockade of its effect at histamine H 2 receptors accounts for several of the world's leading drugs, yet details of the receptor site and mechanism remain elusive. The experimental work of Black et al. [4] and the theoretical gas phase calculations of Weinstein et al. [5,6] have, however, implicated tautomerism in the H a receptor site mechanism. Histamine has two possible tautomers, the proximal (or) and distal (~') forms. Although other species are present in solution, the proposed mechanism involves tautomerism of the amino protonated monocation and free base forms. HC
C/'R
I
(
'
HC
I
C/R
I
N%c/NH
I
HN~c~N
H
H
N(w)H
N(~)H
R = CH2CHzNH 2
Free Base
+
R = CH2CH2NH 3
Monocation
The ratio of tautomers can be described by the equilibrium constant K T = [N(~')H]/[N(~r)H]
(1)
or the corresponding difference in Gibbs free energy, AG T.
Abbreviation: FEP, free energy perturbation. Correspondence: W.G. Richards, Physical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, U.K.
Using the energy cycle below, the calculation of AGT(aq) has two parts to it. First the difference in energy between the tautomers in the gas phase is calculated. The tautomers are then solvated and the difference in free energy of hydration between them is computed using the free energy perturbation (FEP) method [7,81. AGT(g)
N(rr)H (g)
,
N('r)H (g)
AG1 (aq)
N(~')H (aq)
~ N(,r)H (aq)
To calculate AGT(g ), molecular enthalpies at 0 K were calculated using the G A U S S I A N 86 [9] and C A D P A C [10] ab initio programs with various basis sets, and then corrected to Gibbs energies at 298 K using thermodynamic data from A M P A C [11]. Geometries for the ab initio studies were fully optimised up to the 6-31 G level, starting from the known crystal structure of both the histamine base [12] and the monocation [13]. The difference in Gibbs free energy of hydration of the two tautomers was calculated using the FEP module of the A M B E R [14] suite of programs, mutating the Tr tautomer into the z tautomer in solution during the course of a molecular dynamics simulation, and calculating the free energy change. The molecular mechanics parameters for the histamine tautomers were taken from the A M B E R all atom force field [15]. Charges were derived by fitting to the molecular electrostatic potential obtained from the 631G ab initio wavefunction using the program Q U E S T [14]. The starting tautomer, in a box of TIP3P water molecules [16] was first minimized and then equi-
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
159 Table I
The oariation a.u. = 2625.35 in k J . mol - 1, basis sets and
in tautomer ab initio gas phase enthalpies, in a.u., (1 k J . m o l - 1) and the difference in tautomer enthalpy, A H r at 0 K for histamine monocation and base using different geometries
Basis set
A b initio energies in a.u.
A H . r ( g ' 0) ( k J m o l - 1)
N( ~')H
N ( or ) H
a) H i s t a m i n e b a s e RHF/STO-3G//a
- 353.44832
- 353.44968
3.56
RHF/3-21G//b
- 355.90289
- 355.90249
- 1.05
RHF/6-31G//c RHF/6-31G *//c
- 357.74957 - 357.89988
- 357.74828 - 357.89920
- 3.40 - 1.79
RHF/6-31G * *//c
- 357.92232
- 357.92158
- 1.94
MP2/6-31G *//c MP2/6-31G * *//c
- 359.02716 - 359.10541
- 359.02623 - 359.10447
- 2.45 - 2.48
RHF/STO-3G//a
- 353.90906
- 353.89247
- 42.55
RHF/3-21G//b
- 356.29941
- 356.27894
- 53.76
RHF/6-31G//c
- 358.13425
- 358.11319
- 55.30
RHF/6-31G *//c
- 358.28819
- 358.26993
- 47.94
RHF/6-31G * *//c
- 358.31282
- 358.29440
- 48.38
MP2/6-31G *//c MP2/6-31G * *//c
- 359.41381 - 359.49639
- 359.39455 - 359.47718
- 50.56 - 50.42
b) H i s t a m i n e m o n o c a t i o n
Basis set [22-26] n o t a t i o n is e n e r g y calculation m e t h o d / / g e o m e t r y calculation m e t h o d . M o l e c u l a r g e o m e t r y was f o u n d f r o m e n e r g y minim i z a t i o n calculations using a restricted H a r t r e e - F o c k ( R H F ) m e t h o d a n d (a) S T O - 3 G , (b) 3 - 2 1 G or (c) 6-31G. M P 2 shows that the c a l c u l a t i o n was p e r f o r m e d using Moller-Plesset 2nd o r d e r p e r t u r b a tion theory.
librated by molecular dynamics simulation for 4.5 ps. The dynamics were performed using periodic boundary conditions at constant temperature and pressure, using SHAKE [17] to constrain all bonds and a non-bonded cutoff of 0.8 nm (histamine base) or 0.9 nm (histamine monocation). The perturbation was carried out over 21 separate simulations (or windows) with a mutation parameter of AX = 0.05. At each window, 500 steps (1 ps) of equilibration were followed by 2000 steps (4 ps) of data collection. The methods used are reported in more detail in an earlier paper which used the same techniques to calculate the tautomer ratio of 4(5)-methylimidazole in aqueous solution [1]. The results of the ab initio gas phase calculations are shown in Table I. All the basis sets used, with one exception, predict that both the histamine base and the histamine monocation are more stable in the N(~')H tautomer, with the preference being far greater for the monocation. In contrast, the STO-3G result, in agreement with Topiol et al [18], predicts that histamine base is more stable in the N(~r)H form. Table II shows the FEP results, which are the difference in free energy of hydration at 298 K between the tautomers, AAG(hyd) = AG(hyd, 'r) - AG(hyd, rr)
(2)
Taking into account the FEP errors, there is very little difference in the free energies of hydration of the histamine base tautomers, although the N(~')H appears to be slightly more strongly solvated. In contrast hydration favours the N(~r)H histamine monocation over the N(~')H form. Presumably protonation of the side-chain changes the differences in solvation energies due to changing the accessibility of hydrogen bonding sites to the aqueous solvent. The final results (Table III) show the difference in Gibbs free energy between the tautomers in both the gas and solution phases at 298 K. Experimentally determined results are also given for comparison. It can be seen that with good quality basis sets the calculated ratio of histamine base tautomers is in good agreement with the only experimental figure available. The histamine monocation calculation is not in such good agreement with experiment, but it is qualitatively correct: the calculated difference in hydration energy decreases the gas phase energy difference to give a AG v much closer to experiment. Given the good agreement of the histamine base and earlier 4(5)-methylimidazole results [1] seems likely that the most significant error is the underestimation of the coulombic interactions for the monocation by implementing a non-bonded cutoff in the simulations. It may also be significant that the only experiment which measured the ratio in histamine itself, rather than that of the N-methyl derivatives, was that of Wasylishen and Tomlinson [19], who found the ratio to be much more in favour of the N(~-)H tautomer than previous experiments, but were unable to give an exact value. Table II
The F E P results T w o i n d e p e n d e n t F E P r u n s w e r e m a d e on each system. R u n 1 is for the p e r t u r b a t i o n N(~r)H---, N O - ) H ; r u n 2 is for NO-)H---, N ( ~ r ) H . Both these i n d e p e n d e n t p e r t u r b a t i o n s w e r e r u n twice, in the f o r w a r d a n d then the reverse directions. E a c h p e r t u r b a t i o n also c a l c u l a t e s the e n e r g y in b o t h directions. This n u m b e r of s i m u l a t i o n s allows the statistical errors i n v o l v e d in the calculations to be a s c e r t a i n e d * Figures are A A G ( h y d ) in k J . m o 1 - 1 N ( ~ r ) H --} N ( z ) H . Run 1
and
given for the m u t a t i o n
Run 2
a) H i s t a m i n e base Forward - 2.04 Backward - 3.81 Average - 2.93 A A G ( h y d ) = - 1.79 + 1.36 k J-
__.0.67 + 0.35 + 0.76 mol - 1
b) H i s t a m i n e m o n o c a t i o n Forward 32.51 +__0.57 Backward 29.66 + 0.31 Average 31.09 + 0.65 A A G ( h y d ) = 32.71 + 2 . 2 6 k J - m o 1 - 1
- 1.78 + 1.10 0.49 + 0.25 - 0.65 + 1.13
35.76 -t- 0.90 32.88 + 1.97 34.32 + 2.17
* E r r o r in each r u n is the s t a n d a r d d e v i a t i o n b e t w e e n the two energies c a l c u l a t e d d u r i n g the p e r t u r b a t i o n . A v e r a g e errors o v e r the i n d e p e n d e n t runs a n d all r u n s are also s h o w n .
160 Table I1t
Difference in free energies, in kJ. tool 1, for the tautomeric shift N(~r)H --* N('r)H for histamine base and histamine monocation The accuracy of the AG(aq, 298) results are, given the inherent errors in the gas phase calculations, dependant on the statistical errors in the FEP results given in Table 1I. Basis set
Histamine base
Histamine monocation
AG(g, 298) AG(aq, 298) AG(g, 298) AG(aq, 298) (a) Calculated * AM127 RHF/STO-3G//a RHF/3-21G//b RHF/6-31G//c RHF/6-31G*//c RHF/6-31G * * / / c MP2/6-31G * / / c MP2/6-31G * * / / c (b) Experimental * * Charton [28] Ganellin [29] Reynolds [20] Waylishen [19]
**
- 2.17 3.77 - 0.83 -3.18 - 1.57 - 1.71 -2.23 - 2.25
- 3.95 2.00 - 2.61 -4.96 - 3.35 - 3.50 -4.00 - 4.03
--- - 3.4
34.56 - 42.15 - 52.35 -53.89 - 46.53 - 46.97 -49.15 49.02
67.28 - 9.43 - 19.64 -21.18 - 13.82 - 14.26 -16.44 - 16.30
- 2.1 - 3.7 = - 3.4
