Chem. Res. Toxicol. 1989, 2, 144-145
144
Observation of a Substituent Effect on the Stereoselectivity of Glutathione S-Transferase toward Para-Substituted 4-Phenyl-3-buten-2-ones Sir: The glutathione S-transferases (EC 2.5.1.18) are known to catalyze Michael additions of glutathione (GSH) to a,p-unsaturated aldehydes and ketones such as ethacrynic acid, 4-hydroxyalkenals, and trans-4-phenyl-3buten-2-one (1) (1-3). In principle, the enzyme can exhibit a preference for addition of GSH to one of the two prochiral faces of an enone such as 1, giving an excess of one of the two diastereomeric products (Scheme I).l In this paper we report that isoenzyme 4-4 of rat liver GSH transferase exhibits such stereoselective behavior and that its extent depends on the reactivity of the enone. Linear free energy relationships observed for both the kinetics and stereoselectivity of the reaction allow the substituent effects on both diastereomeric transition states to be estimated. The kinetic constants and relative stereoselectivity of the isoenzyme 4-4 catalyzed addition of GSH to substrates 1-52 are easily determined by UV-visible spectroscopy and HPLC.3 The results are summarized in Table I. Although the absolute configurations of the two diastereomers A and B have not yet been determined, it is evident from the circular dichroism spectra of the products that the isomers eluting first (isomers A) on reversed-phase HPLC all have the same c~nfiguration.~ The log of (kc/Km)obsd,which is the rate constant associated with formation of the transition state for the first irreversible step from free enzyme and substrate, is only poorly correlated with the substituent constants u, go,,'u or u- (? between 0.76 and 0.90). However, inclusion of the Hansch hydrophobic substituent constant a (4) by multiple regression analysis leads to an excellent correlation with u (eq 1) with papp = 0.93 and caPp= 0.43, r2 = 0.994, as
1%
(kc/Km)obsd
- cappa = Papp'
"I
I
0
-0.4
0.0
0.4
0.8
sigma Figure 1. Linear free energy relationships for kinetics and stereoselectivity of isoenzyme 4-4 toward 1-5. Lines are fits of the data to the general equation log Y - CT = pa. For log ( k c / K m ) o m (o),pspp = 0.93, c = 0.43, r2 = 0.994; for log (kc/Km)A(@, P A = 0.78, CA = 0.44,?'= 0.976; for log (k,/K,,,)B( O ) , p~ = 1.74, CB = 0.51, ? = 0.994; for log ([A]/[B]) (A),pOm = -0.94, cow= -0.07, r2 = 0.998.
+ flSH\.; Scheme I
X
isozyme 4-4
(1)
shown in Figure 1. Correlations with uo, u+, and u- were also respectable (r2between 0.96 and 0.98). For the situation of two parallel transition states contributing to (kc/Km)obsd, papprepresents a weighted average value for the virtual transition state composed of A* and B* and is dominated by the transition state with the lowest free energy of activation (5). Quantification of the stereoselectivity allows factoring of (kc/Km)obsdinto the rate constants (kc/Km)Aand (kc/Km)Bfor the two parallel reactions. Good correlations with u are obtained with both rate constants (Figure 1) with pA = 0.78, CA = 0.44, and pB = 1.74, cB = 0.51. It is clear from the very different p values that the two diastereomeric transition states differ in The absolute configurations of neither diastereomers A and B nor the corresponding transition states A* and B* should be inferred from Scheme I. Para-substituted trans-4-phenyl-3-buten-2-ones were prepared by Claisen-Schmidt condensation of the corresponding para-substituted benzaldehydes with acetone (8). Isoenzyme 4-4was purified to homogeneity as previously described (9). Initial rates were determined spectrophotometrically at pH 6.5 and 25 OC by using the following As at the given wavelength 1, Acm = -17000 M-' cm-'; 2,Assos = -19400 M-' cm-'; 3,AcSm = -13 300 M-l cm''; 4,Acm = -19500 M-' cm-'; 5, AsBll = -15300 M-' cm-l. Separation of the product diastereomers was accomplished by using a Beckman Ultrapore C8 column (4.6mm X 25 cm) eluted at 0.5 mL/min with 0.1 M ammonium acetate (pH 3.8)containing the following concentrations of CHBOH. Retention times are given in parentheses: 1, 12.5% CHBOH,1A (20.6 min), 1B (22.8min); 2, 12%-50% CH30H in 60 min, 2A (33.1min), 2B (36.6min); 3, 9%-15% CH30H in 60 min, 3A (44.3min), 3B (47.7min); 4, 12.5% CH,OH for 20 min, then to 40% CH30H in 30 min, 4A (41.1 min), 4B (43.0min); 5,same conditions as for 4,5A (44.2min), 5B (48.0 min). The A isomers in all cases exhibited positive CD transitions in the near-UV (240-280nm).
0893-228x/89/2702-0144$01.50/0
electronic character. The transition state (B*) for the reaction which is least effectively catalyzed by the enzyme is considerably more sensitive to the presence of an electron-withdrawing group in the para position. Inasmuch as the log of the ratio of the two products reflects the difference in free energies of activation for the two diastereomeric transition states, it can be readily shown that a linear free energy relationship exists between this quantity and u as expressed in eq 2. Thus the ob(2) log ([A]/[B]) - (cA - C B ) = ~ (PA - PB)Q servation of a substituent effect on the stereoselectivity of the reaction (Figure 1)with p o = ~PA - PB = -0.94 and Cobsd = cA - cB = -0.07 provides an alternative view of the substituent effects on the two parallel transition states. In addition, the rather small influence5 of the hydrophobic term on the stereoselectivity due to the fact that the coefficients cA and cB tend to cancel (eq 2) suggests that its inclusion in the original analysis is warranted. The exact nature of the difference between the two transition states in the enzyme-catalyzed reaction is not understood. We propose here that one orientation of the substrate in the active site of the enzyme, which results in attack of GS- on one prochiral face of the enone, provides for more effective dispersion of charge in the tran-
Correlation coefficient for the stereoselectivity is only marginally improved (0.994to 0.998)by inclusion of the parameter T.
0 1989 American Chemical Society
Communications
Chem. Res. Toxicol., Vol. 2, No. 3, 1989 145
Table I. Kinetic and Stereoselectivity Data for Isoenzyme 4-4 of Rat Liver GSH Transferase with Para-Substituted 4-Phenyl-3-buten-2-ones substrate substituent d irb (ke/K,Jom, M-' 5-l kct s-l mol % stereoisomer A 1 H 0 0.00 (4.8 f 0.2) x 104 8.5 f 0.7 0.90 -0.17 0.56 (5.0 f 0.2) x 104 4.7 f 0.3 0.93 2 CH3 3 OCH3 -0.27 -0.02 (2.0 f 0.1) x 1 0 4 2.2 f 0.1 0.95 4 Br 0.23 0.86 (1.5 f 0.1) X lo5 4.5 f 0.3 0.84 5 NO2 0.78 -0.28 (1.6 0.1) x 105 22.0 f 4.0 0.66
*
Values taken from ref 6. *Values from ref 4.
Kinetic constants were derived from initial rate measurements analyzed by the program
HYPER (7).
sition state than does the other. This particular transition state (A*),which has a lower activation energy, should have a considerably smaller dependence on the nature of the substituent. I t is not unreasonable to suggest that the enzyme may provide for the protonation of the carbonyl group (protonation of the incipient enolate) in this transition-state diastereomer (A') and not in B*. Determination of the absolute configuration of the products is under investigation.
Acknowledgment. This work was supported in part by NIH Grant GM-30910. R.N.A. is the recipient of a NIH Research Career Development Award (ES 00133,1984-89). Support of the Japanese Ministry of Science and Education for the leave of Y.K. from the Department of Chemistry, Shimane University, is gratefully acknowledged.
(3) Danielson, U. H., Esterbauer, H., and Mannervik, B. (1987) Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalyzed by mammalian glutathione transferases. Biochem. J. 247, 707-713. (4) Fujita, T., Iwasa, J., and Hansch, C. (1964) A new substituent constant, 7,derived from partition coefficients. J. Am. Chem. SOC.86, 5175-5183. (5) Schowen, R. L. (1978) In Transition States of Biochemical Processes (Gandour, R. D., and Schowen, R. L., Eds.) pp 77-114, Plenum Press, New York. (6) Hansch, C., and Leo, A. (1979) Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York. (7) Cleland, W. W. (1979) Statistical analysis of enzyme kinetic data. Methods Enzymol. 63, 103-138. (8) Drake, N. L., and Allen, P., Jr. (1948) Benzalacetone. Org. Synth. Collect. Vol. 1, 77-78. (9) Chen, W.-J., Graminski, G. F., and Armstrong, R. N. (1988) Dissection of the catalytic mechanism of isoenzyme 4-4 of glutathione S-transferase with alternative substrates. Biochemistry 27, 647-654.
Yasuo Kubo, Richard N. Armstrong*
References (1) Boyland, E., and Chasseaud, L. F. (1968) Enzymes catalyzing conjugations of glutathione with a,&unsaturated carbonyl compounds. Biochem. J. 109, 651-661. (2) Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) Glutathione S-transferases. The first steD in mercaDturic acid formation. J. Biol. Chem. 249, 7130-7139.
Department of Chemistry and Biochemistry University of Maryland College Park, Maryland 20742 Received February 27, 1989
* Address correspondence to this author.
