Oct 22, 1982 - Eklund, H., Samama, J.-P., Wallen, L., Branden, C.-I.,. Akeson, A. & Jones, T. A. (1981) J. Mol. Biol. 146,. 561-587. Jones, T. A. (1978) J. Appl.
Biochem. J. (1983) 211, 391-396 Printed in Great Britain
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Enantioselective affinity labelling of horse liver alcohol dehydrogenase Correlation of inactivation kinetics with the three-dimensional structure of the enzyme Knut H. DAHL,* Hans EKLUNDt and John S. McKINLEY-McKEE* *Department ofBiochemistry, University of Oslo, P.B. 1041, Blindern, N-Oslo 3, Norway, and tDepartment of Chemistry and Molecular Biology, Swedish University ofAgricultural Sciences, S-750 07 Uppsala 7, Sweden
(Received 22 October 1982/Accepted 30 December 1982) Kinetic data for the inactivation of horse liver alcohol dehydrogenase with S2-chloro-3-(imidazol-5-yl)propionate at pH 8.2 were correlated with the three-dimensional structure of the enzyme. The R-2-chloro-3-(imidazol-5-yl)propionate enantiomer did not inactivate the enzyme, and the reaction is thus enantioselective. Inactivation follows an affinity-labelling mechanism where a reversible complex is formed before the irreversible alkylation and inactivation of the enzyme. A reversible complex is also formed with the non-inactivating enantiomer, and this shows that the selectivity occurs at the irreversible step. By using a computer-controlled display system, models of the two enantiomers of 2-chloro- and 2-bromo-3-(imidazol-5-yl)propionate were built into a model of the enzyme so that the imidazole moiety was liganded to the active-site metal, while the carboxylate group interacted with the general anion-binding site. The conformation of the imidazole derivatives and their orientation in the active site were adjusted to minimize unfavourable steric interactions. It was clear that alkylation of cysteine-46 could proceed with the S-enantiomer bound in this way, but not with the R-enantiomer. Model building thus agrees with the inactivation kinetics and indicates the structural origin of the enantioselectivity. The structure of crystalline horse liver alcohol dehydrogenase is known from X-ray-diffraction studies of the apoenzyme to 0.24nm (2.4A) resolution (Eklund et al., 1976) and of the ternary complex with NADH and dimethyl sulphoxide to 0.29nm (2.9A) resolution (Eklund et al., 1981). Each subunit contains two zinc atoms, one of which, the catalytic zinc, is located at the bottom of a hydrophobic pocket and is part of the active site. The catalytic zinc atom is liganded by the three protein ligands, Cys-46, Cys-174 and His-67, and tetrahedral co-ordination is completed in the enzyme by an H2O/OH- ligand, and in the ternary complexes with a substrate or an effector molecule. The metal-bound thiol group of Cys-46 has been modified with various alkylating agents. With iodoacetate and other halo acids, modification follows an affinity-labelling mechanism, which involves formation of a reversible complex before the irreversible alkylation and inactivation of the enzyme (Reynolds & McKinley-McKee, 1969; Dahl & Abbreviations used: Epps, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid; BIP, 2-bromo-3-(imidazol5-yl)propionic acid; CIP, 2-chloro-3-(imidazol-5-yl)propionic acid.
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McKinley-McKee, 1981a). The reversible complex involves the carboxylate group of the halo acids binding to a general anion-binding site, which normally binds the pyrophosphate group of the coenzyme. 2 - Bromo- 3 - (imidazol - 5 - yl)propionate (BIP) also reacts in accordance with this mechanism, but in this case the reversible complex is in addition stabilized by the imidazole ring binding to the active-site metal atom. So far, it has not been possible to obtain the pure enantiomers of BIP. However, the role of configuration in affinity labelling has been investigated by using the enantiomers of the corresponding compound 2 - chloro - 3 - (imidazol - 5 - yl)propionate (CIP), even though its chemical reactivity is far less than that of BIP (Dahl & McKinley-McKee, 198 lb). The reaction turned out to be totally enantioselective, since S-CIP inactivated the enzyme, whereas with R-CIP no reaction was observed (Dahl et al., 1979). Previously, enzyme kinetic data on the reduction of alkylated cyclohexanones have been correlated with the three-dimensional structure of the enzyme (Dutler & Brinden, 1981). In these studies favourable and unfavourable interactions observed in
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K. H. Dahl, H. Eklund and J. S. McKinley-McKee
models of enzyme-NAD+-alkylcyclohexanol complexes could be correlated with kinetic parameters. The aim of the present work was to investigate the nature of the enantioselective inactivation of liver alcohol dehydrogenase. First, it was necessary to determine whether it is the reversible or the irreversible step in the affinity-labelling mechanism that is responsible for the enantioselectivity. Secondly, model building was used to correlate the data from the inactivation kinetics with the threedimensional structure in order to clarify the structural origin of the enantioselectivity. Materials and methods The source, purity and assay of horse liver alcohol dehydrogenase were as described previously (Reynolds & McKinley-McKee, 1969). Before inactivation, the enzyme was dialysed against two changes of a 100-fold volume of 40mM-Epps/NaOH buffer,
pH18.2. Epps (free acid) and (R,S)-BIP were obtained from Sigma Chemical Co. R- and S-CIP were generously given by Professor H. C. Beyerman, Technische Hogeschool, Delft, The Netherlands. Enzyme inactivations were performed at 23.50C and pH8.2 in 20mM-Epps/NaOH buffer, with an enzyme concentration of about 5,UM. Before use BIP and CIP were titrated to neutrality with alkali. During inactivation, the reaction was followed by withdrawing samples for assay of enzyme activity. Enzyme-inactivation data were processed by a DEC-10 computer by using the program INAKT written by A. Christophersen, which fitted each inactivation curve to the first-order decay curve by non-linear regression. The inactivation rates obtained at various inactivator concentrations were further fitted to the saturation curve, and the kinetic constants with their standard deviation resulted. Semi-logarithmic inactivation plots as well as double-reciprocal plots were produced by a Calcomp plotter connected to the computer. Models of R- and S-BIP (or -CIP) were built into the active site of a refined model of the orthorhombic apoenzyme structure previously determined to 0.24nm resolution (Eklund et al., 1976). Model building was performed on a Vector General 3404 display system controlled by a DEC-PDP1 1/34 computer by using the model-building program FRODO (Jones, 1978, 1982). Stereo diagrams were drawn on a Hewlett-Packard pen-plotter connected to the display system, by using programs written by T. A. Jones.
Results Enzyme inactivation with CIP S-CIP inactivated the enzyme in a reaction that was first-order with respect to enzyme. This is
50 40 40
30
-
-
20
10 I 0
1000
500
2000
1500
Time (min)
Fig. 1. Semi-logarithmic plotsfor the inactivation of liver alcohol dehydrogenase with S-CIP at various concentrations compared with R-CIP (10mM) and a control Enzyme was about 5,M in 20mM-Epps/NaOH buffer, pH 8.2. Concentration of S-CIP: o, 0.25 mM; , 0.37mM; 0, 0.50mM; U, 0.75mM; A, 1.OmM; A, 2.5mM; V, 5.OmM; V, 10.0mM. Iu, R-CIP (10mM); O, control.
5000
4000
-2
-1
0
1
2
3
4
5
1/[S-CIPi (mM-) Fig. 2. Double-reciprocalplotfor the inactivation of liver alcohol dehydrogenase with S-CIP resulting from the inactivation plots in Fig. 1
evident from the semi-logarithmic inactivation plots in Fig. 1. However, with the other enantiomer R-CIP, no reaction was observed and the curve was indistinguishable from the control with no inactivator added. This shows, in agreement with previous results (Dahl et al., 1979), that inactivation with CIP is enantioselective, since inactivation is observed only with the S-enantiomer. The inactivation rates gave a saturation effect with increasing S-CIP, as shown in Fig. 2. The kinetic constants calculated for inactivation with S-CIP are given in Table 1. Previously a minimum inactivation half-time of 915 min and a KI of 1.70mM were observed (Dahl et al., 1979). Phosphate buffer, pH 7.8 and I0. 1, was used previously, and the 1983
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Enantioselective affinity labelling
protecting effect of phosphate anions in the earlier experiments resulted in the higher observed KI value (Dahl & McKinley-McKee, 1980). The Epps buffer used in the present experiments has little interaction with the enzyme at the concentration used (Syvertsen & McKinley-McKee, 1981). Enzyme inactivation with BIP and protection with CIP BIP inactivates liver alcohol dehydrogenase similarly to S-CIP. Inactivation is first-order with respect to enzyme, and a saturation effect is observed with increasing BIP concentration (Dahl & McKinley-McKee, 1981a). Thus inactivation with both compounds follows an affinity-labelling mechanism with a reversible enzyme-inactivator complex formed before the irreversible inactivation. As the KI values in Table 1 show, the affinities of BIP and S-CIP for the enzyme active site are also similar. On the other hand, inactivation with BIP is more than 100 times faster than with CIP, a difference that agrees with the different leavinggroup abilities of chloride and bromide (Dahl & McKinley-McKee, 1981 b). Since CIP is far less Table 1. Kinetic data for enzyme inactivation with BIP and S-CIP In the affinity-labelling mechanism K1
E+I
-
k2
El E.IIE is free enzyme, E I the reversible complex and E' irreversible inactivated enzyme. K1 is the dissociation constant for the reversible complex, and k2 is the rate constant of inactivation. The minimum half-time of inactivation t* minimum = (ln 2)/k2. The values given and their standard deviation result from computation with the program INAKT. Enzyme inactivations were performed in 20mM-Epps/NaOH buffer, pH8.2, at 23.50C.
BIP S-CIP
(mM) (min) 0. 128 + 0.002 5.41 + 0.06 0.69 + 0.02 (9.71 + 0.25)x 10-4 713 + 19 0.73 + 0.06
Table 2. Dissociation constants for R- and S-CIP determined from the degree of protection against the inactivation of liver alcohol dehydrogenase by BIP The K, values were calculated from the ratio of the slopes of the plots in Fig. 3. For both enantiomers concentrations of 1 mm and 2.5 mm were used for
protection. KI (mM)
Concn.
S-CIP R-CIP
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Model-building studies If the enantioselectivity of inactivation with CIP were due to the mode of binding of the two enantiomers in their reversible complexes with the enzyme, this should be evident from the enzyme structure. The availability of the structure data obtained from X-ray-diffraction analysis of the orthorhombic apoenzyme crystals at 0.24nm resolution allowed examination of the structural reason for the enantioselective reaction. The model-building program FRODO (Jones, 1978, 1982), originally designed for building models of macromole-
K1
ti, minimum
(min-')
reactive than BIP, R- and S-CIP can be used for protection of the enzyme against inactivation by BIP. They inhibit inactivation by BIP since they bind reversibly to the same site on the enzyme as BIP does. Even if S-CIP also inactivates the enzyme, this reaction becomes unimportant and can be neglected, since it is much slower than that with BIP. From the degree of protection by R- and S-CIP against BIP, the reversible dissociation constants for the two enantiomers of CIP can be determined, and hence the role of configuration on their binding affinity. This has been performed with two concentrations of R- and S-CIP. The double-reciprocal plots in Fig. 3 show that protection is competitive, and indicate that BIP and CIP have the same binding site. Also, for both concentrations used, protection by S-CIP is greater than by R-CIP. The dissociation constants for R- and S-CIP given in Table 2 are calculated from the ratio of the slopes. The KI values show that the CIP enantiomer that inactivates the enzyme also has the higher affinity.
...
1 mM
0.85 1.09
2.5 mM 0.97 1.18
-2
-1
0
1
2
3
4
5
1/[BIPl (mM-') Fig. 3. Double-reciprocal plots for the inactivation of liver alcohol dehydrogenase with BIP showing the protecting effect of CIP Enzyme was about 5,M in 20mM-Epps/NaOH buffer, pH8.2. Protector: 0, I.OmM-S-CIP; 0, 2.5 mM-S-CIP; A, 1.OmM-R-CIP; v, 2.5 mM-R-CIP. O, Baseline without protector.
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34K. H. Dahl, H. Eklund and J. S. McKinley-McKee
cules from electron-density data, is also appropriate for computer simulation of the binding of smaller molecules to an enzyme molecule for which a structure model already exists. Models of R- and S-BIP were thus built and fitted into the active site of the enzyme model. A model of BIP was constructed by fusing a propionic acid side chain with C-4 (or C-5) in the imidazole ring. In the model building, free rotation was allowed around the following three bonds:
atoms of the imidazole moiety should be liganded to the catalytic zinc atom; (2) the carboxylate group should be directed towards the general anion-binding site so that charge interaction is possible; (3) the electrophilic carbon atom (C(a)) should be-- positioned so that the sulphur atom of Cys-46 can attack from one side while bromide leaves from the other side, in accordance with an SN2 mechanism. This means that Br, C() and SCys-46 are linear.
Ccarboxy-C(a)-C(MCring. Previously it had been concluded that BIP binds reversibly to the active site of liver alcohol dehydrogenase by one of the imidazole nitrogen atoms liganding to the active-site metal, while the carboxylic group is orientated towards the general anion-binding site, implicated as Arg-47 (Dahl & McKinley-McKee, 198 la). The design of the reversible enzyme-BIP complex was thus based on the following considerations: (1) one of the nitrogen
Description of the reversible complexes For both enantiomers of BIP, it was difficult to find the best conformation that would fit into the active-site pocket, since this was narrow, with little freedom for movement. When the BIP molecule was bound to the enzyme with N-1 or -r bound to the metal atom, the possible movement of the imidazole ring is restricted by the metal atom and Cys-46 in such a way that C(.) could not be brought into van der Waals contact with SCys-46* Thus the close
(b)
Fig. 4. Stereo diagram of the active-site region of liver alcohol dehydrogenase showing the probable reversible complexes formed with (a) S-BIP and (b) R-BIP
1983
Enantioselective affinity labelling proximity necessary for nucleophilic attack by cysteine sulphur on C(,) could be excluded. However, this was possible when the other nitrogen atom of the imidazole ring was bound to the catalytic zinc atom. It is thus concluded that N-3 or -7x in the imidazole ring ligands to the metal atom. When the imidazole ring was positioned in this way, the carboxylate group of BIP could be positioned appropriately for charge interaction with Arg-47 and Arg-369. The side chain of Arg-47 is flexible in the apoenzyme (Eklund et al., 1976) and has no fixed position in the original model, but is easily moved so as to interact. For the complex with S-BIP, the model shown in Fig. 4(a) satisfied all the above considerations. Here N-3 of imidazole is liganded to zinc, the Zn-N distance being 0.215 nm (2.15 A). The carboxylate group is directed towards Arg-47 and Arg-369, at a distance suitable for charge interaction. At the same time, C(a) is positioned appropriately for nucleophilic attack from SCys-469 with the angles between the Br-C(a) bond and a possible C(,)-S bond being 1610. In this reversible complex, SCyS 46 is in van der Waals contact with the C(a)) C¢o) and C carboxy carbon atoms. It is readily apparent that S-BIP in this reversible complex can alkylate Cys-46 as a result of nucleophilic attack by sulphur followed by the leaving of bromide from the opposite side. With R-BIP, however, it was not possible to design a complex with imidazole on zinc, carboxylate towards the general anion-binding site and the electrophilic carbon atom positioned appropriately for alkylation. Nevertheless, a probable complex can be designed with imidazole bound to the zinc atom and the carboxylate group pointing towards the two arginine residues, similar to that with the S-enantiomer. In this reversible complex, which is shown in Fig. 4(b), the sulphur atom is in van der Waals contact with C'carboxy' C(a), C(A) and Br. Here attack on the electrophilic carbon by sulphur is from the wrong direction, since the angle between the Br-C(a) bond and a possible C(a)-S bond is about 800, which makes alkylation unlikely. The nucleophile and the leaving group come too close to each other to allow a nucleophilic attack. If the bromine and the carboxyl group of BIP are turned around the C(a-C(A) bond so that the bromine atom is pointing away from the sulphur atom, the carboxylate group comes too close to the protein. Model building thus indicates that both enantiomers can form reversible complexes, but irreversible alkylation of Cys-46 is possible only with the S-enantiomer. Discussion The inactivation of liver alcohol dehydrogenase with CIP is an enantioselective reaction, since only Vol. 211
395 the S-enantiomer inactivates the enzyme. The present results agree with previous results (Dahl et al., 1979), but KI determined in the present work is more meaningful since Epps buffer is used in place of phosphate, which binds to the general anionbinding site and protects the enzyme against inactivation (Dahl & McKinley-McKee, 1980). Generally, the specificity of an enzyme for a substrate is determined by the factor kcat./Km. In the present case, where CIP and BIP mimic substrates, specificity is by analogy expressed by k+2/KI. It is thus of interest to know whether the selectivity for S-CIP is a result of a low KI or a high k+2 compared with the other enantiomer. By using R- and S-CIP for protection of the enzyme against BIP inactivation, this question could be answered. It turned out that both enantiomers of CIP protected the enzyme against BIP, but the S-enantiomer protected somewhat more and had the higher affinity. The difference in affinity is, however, small (Table 2), and does not account for the enantioselectivity in the inactivation reaction. Selection between R- and S-CIP is thus seen to be at the k+2 step, the irreversible reaction. It represents the different orientations of the two enantiomers in the reversible complexes. The S-enantiomer is positioned so that irreversible alkylation can take place, whereas the R-enantiomer is not. The following scheme applies: S-CIP Et E E*S-CIP k-2 R-CIP
1; E *R-CIP
where E is free enzyme, E. reversibly bound enzyme and E' irreversibly inactivated enzyme. A possible cause for enantioselectivity could be different chemical reactivities of R- and S-CIP. However, this is not the case, since the two enantiomers have similar reactivities when their reactions with the thiolate anion of cysteine are measured (Dahl & McKinley-McKee, 1981 b). The observed enantioselectivity of CIP against the enzyme can also be expected for BIP, even if this has not been examined because BIP is available only as a racemic mixture. This means that, when the enzyme is inactivated with BIP, it is only the S-enantiomer that inactivates, while the R-enantiomer inhibits this inactivation and protects the enzyme. The situation is analogous to that where a constant proportion of an inhibitor is present in a substrate, so that with increasing substrate concentration the concentration of the inhibitor also increases in parallel (Dixon & Webb, 1979). If the values of KI for R- and S-BIP are similar, like those
396 for CIP, the consequence is that the observed k+2 (Table 1) will be only about half the true k+2 for pure S-BIP. With regard to KI, the observed value will be about the same as that for pure S-BIP. The structure of the reversible complexes with Rand S-BIP is seen to determine the enantioselectivity. This means that the reacting atoms in the E * S-BIP complex are positioned so that reaction is possible, whereas this is not the case with the E R-BIP complex. Generally this type of reaction follows an SN2 mechanism, and this should occur with the present reaction within the reversible complex. In such a case, the nucleophilic sulphur atom of Cys-46 will attack the electrophilic carbon atom from one side while bromide will leave from the opposite side. In the model building BIP was thus orientated to that the Br-C(a)-SCys-46 centres were linear. The model building agreed with the structure of the reversible complex as predicted from the inactivation kinetics. S-BIP fitted into a position where alkylation was possible, whereas this was not the case with R-BIP. When BIP is bound reversibly in the enzyme active site, it is bound both to the catalytic metal atom and to the general anionbinding site. Being anchored at both ends, the position of BIP is rather fixed. This fixed orientation is responsible for the absolute enantioselectivity of the inactivation. In the present correlation study, where kinetic data are compared with the structure of a reversible complex, the question arises as to how well the models depict the real complexes. Even if BIP is fixed in the reversible complex, there has to be the possibility of some dynamic movement. However, the models in Figs. 4(a) and 4(b) show a static state. These thus only reflect a situation where BIP is likely to be bound. In the case of S-BIP, where the atoms
K. H. Dahl, H. Eklund and J. S. McKinley-McKee
Br-C(a)-SCyS 46 lie on a straight line, Fig. 4(a) represents a complex preceding the transition state in the alkylation reaction. The results obtained by correlating model building and inactivation kinetics are mutually confirmatory. They illustrate how orientation of a substrate in the active site is so crucial for the value of k,2 that absolute enantioselectivity results. This work was supported by the Norwegian Council for Science and the Humanities (N.A.V.F.) and the Swedish Natural Science Research Council. References Dahl, K. H. & McKinley-McKee, J. S. (1980) Eur. J. Biochem. 103,47-51 Dahl, K. H. & McKinley-McKee, J. S. (1981a) Eur. J. Biochem. 118, 507-5 13 Dahl, K. H. & McKinley-McKee, J. S. (1981b) Bioorg. Chem. 10, 329-341 Dahl, K. H., McKinley-McKee, J. S., Beyerman, H. C. & Noordam, A. (1979) FEBS Lett. 99, 313-316 Dixon, M. & Webb, E. C. (1979) Enzymes, 3rd edn., pp. 355-357, Longman, London Dutler, H. & Brand6n, C.-I. (1981) Bioorg. Chem. 10, 1-13 Eklund, H., Nordstr6m, B., Zeppezauer, E., Soderlund, G., Ohlsson, I., Boiwe, T., S6derberg, B.-O., Tapia, O., Branden, C.-I. & Akeson A. (1976) J. Mol. Biol. 102, 27-59 l Eklund, H., Samama, J.-P., Wallen, L., Branden, C.-I., Akeson, A. & Jones, T. A. (1981) J. Mol. Biol. 146, 561-587 Jones, T. A. (1978) J. Appl. Crystallogr. 11, 268-272 Jones, T. A. (1982) in Computational Crystallography (Sayre, D., ed.), pp. 303-317, Oxford University Press, Oxford Reynolds, C. H. & McKinley-McKee, J. S. (1969) Eur. J. Biochem. 10,474-478 Syvertsen, C. & McKinley-McKee, J. S. (1981) Eur. J. Biochem. 117,165-170
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