J. theor. Biol. (1979) 78, 393-403

A Common Active Site Model for Catalysis by Chorismate Mutase-Prephenate Dehydrogenase P. R. ANDREWS AND ELIZABETH

HEYDE

Departments @‘Physical Biochemistry and Biochemistry, The John Curtin School qf‘ Medical Research. .4ustralian National University, Canberra, ‘4.C. T. 3601, .4usrralia (Received 16 .4ugusr 1978, and in reketl,ftirm

8 December 1978)

Comparison of the calculated structures for the transition states of the two reactions catalysed by chorismate mutase prephenate dehydrogenase suggests that both reactions could be catalysed at a common active site. Kinetic data for the enzyme from Arrohacfrr aerogenes are consistent with this possibility. On the basis of these theoretical and experimental data a model for a common active site is developed. In the model, the transition state for each reaction is bound to the enzyme via both of the two substrate carboxyl groups, and can also interact with the coenzyme nicotinamide adenine dinucleotide through a hydrogen bond between the amide moiety of the nicotinamide ring and the hydroxyl group of the substrate. Chorismate, prephenate and 4-hydroxyphenylpyruvate in their ground states form the same hydrogen bond to the coenzyme, but are bound to the enzyme via a single carboxyl group only. The additional bond formed between the enzyme and the transition state structures thus provides the transition state stabilization required for catalysis of both reactions.

1. Introduction mutase and prephenate dehydrogenase exist as separateenzymes in some species of microorganisms and plants, but in several bacterial species, including Aerobacter aerogenes, Escherichia coli and Salmonella t~~phimurium,the two activities are found in association on the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase (Kirschner & Bisswanger, 1976; Pittard & Gibson, 1970). Indeed, chemical inactivation and other studies on chorismate mutaseeprephenate dehydrogenase from A. aerogenesand E. co/i are consistent with a model in which the mutase and dehydrogenase sites are at least close to one another and possibly overlap (Koch, Shaw & Gibson, 1972). Kinetic studies on the enzyme from A. arrogenes indicate that both reactions could occur at a single active site or at Chorismate

393

0071- 5193/79/l 10393+ I1 $02.00/0

I’m1979 Academic Press Inc. (London) Ltd.

P.K.ANDREWS

394

4NDE.HEYDE

two sites with similar kinetic properties (Heyde Xc Morrison. 197X, and in prep. 1. It is possible that these alternative models could be distinguished by comparison of the two transition state structures, which may reasonably be regarded as templates for their respective catalytic sites. Major structural differences between the transition states would thus rule out a common active site model, for which the two transition state structures should be closely related. Although the transition states themselves are not experimentally accessible, detailed geometries for both transition state structures have been calculated in the course of molecular orbital studies (Andrews & Haddon, submitted) of the isomerization of chorismate to prephenate [Fig. l(a)] and the dehydrogenation of prephenate to 4hydroxyphenylpyruvate [Fig. l(b)] ; additional structural evidence concerning the transition state for the mutase reaction has been obtained from studies of transition state analogues (Andrews et LIP.,1977). The present work combines these structural data with the steady-state kinetic evidence to provide a self-consistent single site model for the reactions catalysed by chorismate mutase -prephenate dehydrogenase. 2. Methods Computer programs were written to allow manipulation, comparison and three-dimensional display of molecular structures. The common active site model was derived by stereoscopic superimposition of the hypothetical active coo-

OH

coo-

(bl

coo,a

0 t NAD+

+

OH OH

FIG. 1. (a) The isomerization prephenate to 4-hydroxyphenylpyruvate.

of chorismate

to prephenate.

(b) The dehydrogenation

of

395 sites corresponding to the individual transition state structures. This was achieved by minimizing the sum of the distances between corresponding atoms in the two active sites with respect to the six degrees of intermolecular rotational and translational freedom as well as rotation around low energy bonds, e.g. ring-hydroxyl, ring-carboxyl. The orientation of NAD was then varied to optimize intermolecular hydrogen bonding and hydride ion transfer. The transition state structures were calculated using the MIND013 molecular orbital method (Bingham et al., 1975). CHORISMATE

MUTASE-PREPHENATE

3. Comparison

of Transition

DEHYDROGENASE

States

The substituents of the transition states which are capable of forming strong bonds to the enzyme are the same for both reactions. They are the two carboxyl groups, which are both negatively charged at physiological pH, and the hydroxyl group, which may participate in hydrogen bonding. In comparing the two transition state structures the disposition of these three substituents is therefore of major importance. The transition state for the isomerization of chorismate could, in principle, adopt either a chair-like or a boat-like conformation, but inhibition studies with analogues of both structures indicate that the enzyme-catalysed reaction proceeds via the chair-like intermediate (Andrews rt al.. 1977). The detailed geometry of this structure calculated using MIND0/3 is shown in Fig. 2(a). (a)

(b)

FIG. 2. Stereoscopic drawings of the transition states for (a) the isomerization of chorismate to prephenate, (b) the dehydrogenation of prephenate to 4-hydroxyphenylpyruvate. Oxygen atoms are shaded.

P K. ANDREW!3 AND E. HEYDE 396 It is noteworthy that the transition state is asymmetric: the making bond (dashed line) is some 0.4A longer than a normal C-C bond, while the breaking bond is only 0.1 A longer than the usual C--O bond length. The transition state structure for the dehydrogenation of prephenate to 4hydroxyphenylpyruvate is illustrated in Fig. 2(b). The reaction is partially concerted, with the ring carboxyl group almost detached in the transition state structure (C C bond length c. 4 A), and the breaking C-H bond considerably longer than usual (1.95 A, cf. 1.09 A). A striking feature of these two structures is the marked similarity in both the positions and the orientations of the three potential binding groups, i.e. the ring and sidechain carboxyl groups and the hydroxyl moiety. These similarities, which are evident from Fig. 2, are further emphasized if the two transition state structures are used as templates to provide approximate outlines of the corresponding active sites.

4. Superimposition

of Active Sites

Approximate outlines of the active sites were obtained by assigning appropriate lengths to the bonds between the three transition state substituents and their proposed binding sites on the enzyme. We chose to use an average hydrogen bond length of 2.8 A (Pimental & McLellan, 1960) for the distance between the oxygen atoms of each substituent and the corresponding binding group on the enzyme. In the case of the two carboxyl moieties, the binding group was placed collinearly with the C-CO, bond. For the hydroxyl groups it was either collinear with the O-H bond, as in an O-H -. -X hydrogen bond, or at a torsion angle of 180” to the O-H bond, as in an 0. ~ H- X hydrogen bond. The two hypothetical active sites were superimposed as described in the Methods section. The three binding points obtained are illustrated schematically as triangles in Fig. 3 : it is clear that the optimum disposition of active site binding groups is very similar for the two transition states. An active site suitable for catalysis of both reactions may therefore be obtained by placing suitable binding groups at the intermediate positions shown in Fig. 3. This rudimentary model provides the basis for our proposed active site, which we shall quantify with kinetic data. 5. Kinetic Data and Free Energies of Interaction

The kinetic data obtained for the enzyme from A. uerogenes are summarized in Table 1. These come from steady-state investigations (Heyde & Morrison, 1978) which were qualitatively and quantitatively consistent with a rapid equilibrium random mechanism for the dehydrogenase reaction. For such a

CHORISMATE

MUTASE-PREPHENATE

DEHYDROGENASE

hydroxyl

397

side chom corbcnyl

FIG. 3. A planar representation of the optimal placement of binding groups in hypothetical active sites catalysing the isomerization of chorismate to prephenate (- - --) and the ). Solid circles indicate dehydrogenation of prephenate to 4-hydroxyphenylpyruvate (the intermediate location of binding groups in a possible single site model.

mechanism, the rate of conversion of the central ternary complex to form products is sufficiently slow compared with the rates of all other steps that the interactions of reactants with the enzyme occur under rapid equilibrium conditions; hence all kinetic constants associated with the dehydrogenase reactants are dissociation constants. The mutase reaction may also occur under rapid equilibrium conditions, in which case the value determined for the Michaelis constant for chorismate is a dissociation constant. A dissociation constant for prephenate is also determined from the mutase reaction (Heyde & Morrison, 1978). The free energies of interaction corresponding to these dissociation constants have been calculated from the relationship AC = - RT In K at 30°C. These and certain derived values are recorded in Tables 1 and 2. (A)

ENZYME-SUBSTRATE

INTERACTIONS

The free energies of interaction indicate that chorismate, prephenate and 4-hydroxyphenylpyruvate are each bound to the enzyme with a bond strength of approximately 5 kcal mol-‘. This is consistent with the formation of a single ionic bond (Albert, 1968) involving either one of the two car-boxy1 groups. The ring carboxyl, which remains almost stationary relative to the body of the molecule throughout the mutase reaction, is the more likely candidate, since attachment of the sidechain carboxyl group would require the whole ring system to reorient within the active site. Alternatively, chorismate and prephenate may be bound to the enzyme through different carboxyl groups.

398

P. R. .ANDREWS

AND TABLE

E

HEYDE

1

Dissociation Reactant Chorismate

Prephenate

Reaction Enzyme Enzyme Enzyme Enzymet Enzyme: Enzymes Enzyme

with

COnStant

0-l’

(mM)

*041

Free energy of association (kcal mol

NAD+ NADH

0~016+0GO3 0442 + 0.01 1

- 5.4 - 6.6 - 6.1

NAD’ NADH

0.17 *002 0.21 kO.01 0,030 * 0,003 O@l6+0~010

- 5.2 -5.1 - 6.3 - 6.0

NAD+

044 +047 1.5 10.2 0.032 * om2

-4.7 -3.9 - 6.2 - 4.3 - 4.5 -5.5 -5.6

4WdroxyphenyL pyruvate

Enzyme? Enzyme1 Enzyme

NAD+

Enzymet Enzyme: Enzyme-prephenate Enzyme chorismate Enzyme-hydroxyphenylpyruvate

0.72 0.60 0.11 0.096

0.052 * 0.011

-5.9

NADH

Enzymet Enzymef Enzyme Enzyme

0.086 0.17 0.023 0.059

i 0.015 kO.03 f 0002 * om9

- 5.6 - 5.2 -6.4 - 5.9

2.8 1,7

kO.4 fO.3

-3.5 -3.8

AMP

t Measurement $ Measurement

prephenate chorismate

Enzyme? Enzyme-prephenate

kO.09 kO.16 *0.01 k 04 13

’)

based on dehydrogenase reaction based on mutase reaction

In the dehydrogenase reaction the ring carboxyl is removed to yield 4hydroxyphenylpyruvate, which is clearly attached to the enzyme through the sidechain carboxyl group. (B)

COENZYME-ENZYME

INTERACTIONS

Binding of NAD to the enzyme occurs primarily through the AMP portion of the molecule (3.5 kcal mol-‘, Table 2) with only a limited contribution coming from the nicotinamide ring. The latter is marginally stronger for the puckered reduced form, possibly as a result of coulombic repulsion between the active site and the positively charged ring in the oxidized molecule.

CHORISMATE

MUTASE-PREPHENATE TABLE

DEHYDROGENASE

399

2

Free energiesof interaction between reactants and enzyme at 30°C Interactton ~~~-.-

with

Enzyme+ Reactant

(kcal mol

Chorismate Prephenate 4-Hydroxyphenylpyruvate NAD’ AMP t The values for interaction values (Heyde & Morrison, NAD’ are calculated from interaction for the enzyme complexes.

(C)

--

NAD+ ’)

t

1.7 1.1 1.5

5.4 5.2 4.3 4.4 3.5

with the enzyme are direct experimental 19781, while those for interaction with the difference between the free energies of NADf and (enzyme--substrate)-NAD+

COENZYME-SUBSTRATE

INTERACTIONS

Prephenate binding is enhanced by NAD, independently of oxidation state, but not by AMP, and the binding of both chorismate and 4hydroxyphenylpyruvate is enhanced to a similar extent by the presence of NAD. This enhancement may be due to a direct interaction between the reactants, or to a change in the binding capacity of the enzyme mediated by the NAD (e.g. a conformational change). We favour the first alternative, however, since we know that prephenate and NAD+ must interact for subsequent hydrogen transfer. This being the case, the absence of any effect of AMP on prephenate binding suggests that the substrate-NAD interaction involves the nicotinamide ring, and the failure of the oxidation state of NAD to influence the interaction indicates that the amide sidechain is involved. The most probable interaction is therefore a hydrogen bond, which is consistent with the bond strength of approximately 1.5 kcal mol-’ (Table 2). In view of the probable site of hydrogen transfer from the Cposition of prephenate to the nicotinamide ring, it is likely that the hydrogen bond formed is to the hydroxyl group of prephenate. Two alternative models are possible for this bond, either -N-H- -0- [Fig. 4(a)] or =0---H-O [Fig. 4(b)]. Both models are consistent with the kinetic data, but the former is a marginally stronger bond.

400

FIG. 4. Two alternative sidechain of NAD and O-H---O; (b) N-H---O.

P.R.ANDREWSANDE.HEYDE

configurations the hydroxyl

for the proposed hydrogen bond between group of the dehydrogenase transition

the amide state. (a)

6. Common Active Site Model

According to the preceding analysis, each of the three reactants will be bound to the enzyme-coenzyme complex through a single carboxyl group and the hydroxyl moiety. These two bonds will have no direct catalytic effect, since they stabilize the substrates, transition states and products to the same extent. Thus, for example, the presence or absence of NAD, which is required as a reactant for the dehydrogenase reaction, should not influence the rate of the mutase reaction, but will simply increase the affinity of chorismate, prephenate and the mutase transition state for the enzyme. This effect is observed experimentally (Heyde & Morrison, 1978). The additional binding of the transition states, which is necessary for catalysis, must therefore involve an interaction between the active site and the second carboxyl group in the two transition states. Such an interaction is not possible for any of the three substrates or products, whose substituents are not suitably located to allow binding to all three positions shown in Fig. 3. These effects are illustrated in Fig. 5, where the entire reaction sequence is shown at an active site based on Fig. 3. It includes two positively charged groups, which form ionic bonds with the carboxyl substituents, and the nicotinamide ring of NAD. The latter is positioned to allow hydrogen bonding between the amide and hydroxyl groups, as well as hydrogen transfer from prephenate to nicotinamide. The interactions between the common active site and all three binding groups in the transition states, but not in the substrates or products, are

CHORISMATE

MUTASE-PREPHENATE

DEHYDROGENASE

401

(a)

(b)

(cl

(d)

(e)

FIG. 5. Stereoscopic drawings of the proposed reaction sequence. Catalysis occurs at a single active site represented by the nicotinamide moiety of the coenzyme NAD (lower foreground) and two unidentified centres (8) capable of binding to the substrate carboxyl groups. Light and dark shadings represent the oxygen and nitrogen atoms respectively. (a) Chorismate, (b) mutase transition state, (c) prephenate, (d) dehydrogenase transition state, (e) 4hydroxyphenylpyruvate.

evident in Fig. 5. The extent of stabilization due to these interactions is given in Table 3. The catalysis due to transition state stabilization may then be estimated by comparing the stabilization of the transition state with that of the substrates. For the mutase reaction, which is unimolecular, this reduction in enthalpy of activation provides the major catalytic effect, and is estimated to be in the range 5-10 kcal mol- ‘, corresponding to the strength of a reinforced ionic bond of the type which could be formed by the sidechain carboxyl group (Albert, 1968). This is in excellent agreement with the

402

P. R. ANDREWS

AND

TABLE

E. HEYDE

3

Free energies of inteructions hetN,ern reactant shtituer~ts und the enzywle coenzyme complex for proposed single active site model (lid molt ’ ) Binding Ring carboxyl

Reactant Chorismate Mutase

group

Sidechain carboxyl

1.2:

54t transition

state

Prephenate Dehydrogenase state

-51;

Hydroxyl

5 10s

5.2

-1% 1.1:

Total 6.6 11 16 6.3

transition

4-Hydroxyphenylpyruvate

5 10s

-54 4.3t

-1s 1.5:

t Experimental value $ Value calculated by difference between the free energies of interaction coenzyme and (enzyme reactant) coenzyme complex 4 Values based on the active site model F Range of values for an ionic or reinforced ionic bond (Albert. 1968)

II-16 5.8

for the enzyme

experimental reduction in enthalpy of activation (4-9 kcal/mol) obtained by comparison of the enzymatic and non-enzymatic rates of isomerization (Andrews et al., 1973). For the dehydrogenase reaction a similar reduction in the enthalpy of activation is expected from transition state stabilization. In addition, however, a substantial reduction in the entropy of activation will result from the approximation of prephenate and NAD+ at the active site, especially in view of the proposed hydrogen bond formation between the two substrates. As illustrated in Fig. 5, the formation of this hydrogen bond is consistent with particularly facile hydrogen transfer from prephenate to NAD+, and may play a major role in catalysis. 7. Discussion

The common active site model illustrated in Fig. 5 provides a basic mechanism of catalysis for both reactions catalysed by chorismate mutaseprephenate dehydrogenase, but a number of structural details remain to be established. These include the nature of the interaction between prephenate and NAD+, which could yet prove to be mediated by a conformational change in the enzyme rather than either of the proposed hydrogen bonds (Fig. 4). We hope to define this interaction using NAD analogues in which the amide sidechain is either modified or removed.

CHORISMATE

MUTASE-PREPHENATE

DEHYDROGENASE

403

The identities of the two positively charged groups in the active site are also unknown, although parallel inactivation of both the mutase and dehydrogenase by pyridoxal phosphate has been observed, indicating that a lysine residue may be involved. This would be consistent with the observation that a lysine residue plays a vital role in the mutase site of the mutase-prephenate dehydratase closely related enzyme, chorismate (Gething & Davidson, 1977). Current investigations of the effect of pH on the reactions may settle this question. There is evidence that chorismate mutase-prephenate dehydrogenase from either A. aerogenes or E. coli consists of a single type of polypeptide chain (Koch, Shaw & Gibson, 1970, 1971). For multifunctional enzymes of this kind, the limited information available indicates that different regions of the polypeptide chain are in general responsible for the different activities, although interaction between the regions often occurs (Kirschner & Bisswanger, 1976). Our results suggest that chorismate mutase-prephenate dehydrogenase may differ from this general pattern in containing only one active centre. Such a model is consistent with all of the available kinetic and structural data. REFERENCES ALBERT. A. (1968). Selective To\-icity. 4th edn. London: Methuen. ANDREWS. P. R., CAIN, E. N., RICCARDO. E. & SMITH, G. D. (1977). Biochemistry 16, 4848. ANDREWS, P. R., SMITH, G. D. & YOUNG, I. G. (1973). Biochemistry 12, 3492. BINGHAM, R. C., DEWAR, M. J. S. & Lo, D. H. (1975). J. Am. Chem. Sot. 97, 1285. GETHING, M.-J. H. & DAVIDSON, B. E. (1977). Eur. J. Biochem. 78. 111. HEYDE, E. & MORRISON, J. F. (1978). Biochemiwy 17, 1573. KIRSCHNER. K. & BISSWANGER, H. (1976). Ann. Rev. Biochem. 45, 143. KOCH, G. L. E., SHAW, D. C. & GIBSON, F. (1970). Biochim. Biophy. Ac,ra 212, 387. KOCH, G. L. E., SHAW, D. C. & GIBSON, F. (1971). Biochim. Bi0phy.s. Ac,/a 229, 805. Kocn, G. L. E., SHAW, D. C. & GIBWN, F. (1972). Bioc,him. Biophy. Acra 258, 719. PIMENTAL. G. C. & MCCLELLAN, A. L. (1960). The Hydrogen Bond. San Francisco. London: Freeman. PIITARD. J. & GIBSON, F. (1970). Current Topics in Cellular Regularion 2. ‘9.