The Hard-Soft Acid-Base Principle in Enzymatic Catalysis: Dual Reactivity of Phosphoenolpyruvate YAN LI and JEREMY N

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4612-4616, May 1996 Biochemistry

The hard-soft acid-base principle in enzymatic catalysis: Dual reactivity of phosphoenolpyruvate YAN LI AND JEREMY N. S. EVANSt Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660 Communicated by R. G. Pearson, University of California, Santa Barbara, CA, January 11, 1996 (received for review October 9, 1995)

ABSTRACT In this paper, the chemical reactivity of C3 of Fukui function f(r) (9) is one such quantity that is related to phosphoenolpyruvate (PEP) has been analyzed in terms of the electronic density in the frontier molecular orbitals and density functional theory quantified through quantum chem- thus determines the chemical selectivity (10). For a chemical istry calculations. PEP is involved in a number of important reaction, a Taylor series expansion can be performed up to enzymatic reactions, in which its C3 atom behaves like a base. second order by energy perturbation methods within the In three different enzymatic reactions analyzed here, C3 framework of density functional theory (11). The energy sometimes behaves like a soft base and sometimes behaves like change for a molecule will be, to second order in the changes a hard base in terms ofthe hard-soft acid-base principle. This AN and Av(r): dual nature of C3 of PEP was found to be related to the conformational change of the molecule. This leads to a ( aE\- aE testable hypothesis: that PEP adopts particular conforma- AE = AN + tions in the enzyme-substrate complexes of different PEP- aN\ vN,(r) a v (r) N Av (r)dr using enzymes, and that the enzymes control the reactivity through controlling the dihedral angle between the carboxy- _E - l( d2E late and the C=C double bond of PEP. + II ) ANAv(r)dr + 2kd2N) ANN2 aNav(r) 2aNv(r) The chemical reactivity of the C==C double bond in phosphoenolpyruvate (PEP) in enzymatic reactions is somewhat of +1 2E an enigma. There are at least three enzymatic reactions in +2 tav(r)av(r') N Av(r)/Av(r')drdr' which PEP participates by providing C3 of its enolpyruvyl moiety rather than its phosphate group (Fig. 1). In the reaction catalyzed by EPSP synthase (1, 2) (Fig. 1, path I), C3 of PEP = ,AN + hAN2 + fp(r)Av(r)dr + ANJf(r)Av(r)dr is protonated during initiation of the reaction, which implies, in terms of the hard-soft acid-base (HSAB) theory, that this carbon is a hard base (because a proton is believed to be a hard acid). On the other hand, in the reaction catalyzed by KD08P 2J av(r') A (r) v (r')drdr' [1] synthase (3, 4) (Fig. 1, path II), this same carbon acts as a soft v A base and is used to attack an aldehyde carbon, which is believed to be a soft base, of the cosubstrate of the enzyme. This dual where r and r' are independent variables of position, r1 is the reactivity of C3 of PEP has nothing to do with entropy; nor can global hardness, and s(r) is the local softness that is related to it be explained in terms of other current theories of enzymatic the Fukui function by s(r) = f(r)/iq. After some mathematical catalysis in a straightforward way. For example, although the manipulations, Eq. 1 can be rewritten as: enzyme active site can determine the relative orientation of the two bound cosubstrates with respect to one another, the AE = + relative binding geometry is unlikely to be sufficient to explain puAN -qAN2 + f p(r)Av(r)dr + ANJf(r)Av(r)dr the dual reactivity of PEP. However, it can be understood in terms of HSAB theory. This paper addresses this issue by using the methods of density functional theory quantified by quan- + a (r)dr) s(r)(Av(r))2dr [2] tum chemistry calculations. 2-(s(r)Av 2

THEORETICAL where a is a positive constant. For a bimolecular reaction of BACKGROUND molecules i andj, the energy perturbations of the whole system The HSAB principle was proposed more than 30 years ago by is AEi + AEj and can be rewritten as: Pearson (5, 6) to predict the stability and reactivity of a generalized acid-base reaction. According to the HSAB prin- AEtotal = AEi + AEj ciple, acids (and bases) can be classified into two kinds: soft and hard. The HSAB principle states that a hard acid likes to react with a hard base and a soft acid likes to react with a soft = (gi - gj)AN + (hqi + -1j)AN2 + f[pi(r)Avi(r) base. Parr et al. (7, 8) have used density functional theory to provide a theoretical basis for the HSAB principle and a method to quantify it. + pj(r)Avj(r)]dr + ANJWVi(r)Avi(r) To study chemical reactivity and selectivity, it is desirable to investigate quantities that characterize local properties. The Abbreviations: DAHP, 3-deoxy-D-arabinoheptulosonate-7-phosphate; EPSP, 5-enolpyruvyl-shikimate-3-phosphate; HOMO, highest occu- The publication costs of this article were defrayed in part by page charge pied molecular orbital; HSAB, hard-soft acid-base; KD08P, 3-deoxy- payment. This article must therefore be hereby marked "advertisement" in D-manno-2-octulosonate-8-phosphate; PEP, phosphoenolpyruvate. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

4612 Downloaded by guest on September 26, 2021 Biochemistry: Li and Evans Proc. Natl. Acad. Sci. USA 93 (1996) 4613 According to Eq. 7, for a given B, the larger local softnesses ia - fj(r)Avj(r)]dr + si(r)Avi(r)dr are, the lower the reaction energy will be; in other words, soft likes soft. Therefore, chemical selectivity and reactivity are controlled by the HSAB principle (6, 8). According to Eq. 5, for a protonation reaction, a minimal s(r) site is favored (11). + lj (f sj (r)A vj (r)dr) - af [si(r)(A vi(r))2 Thus, the match of the Fukui function is important for a chemical reaction to occur via a lower activation energy pathway. + sj(r)(Avj(r))2]dr [3] COMPUTATIONAL METHODS with All the calculations were carried out using the ab initio quantum chemistry program, GAMESS (12). The Fukui function AN = ANi = 2 0 [4] -ANj is approximated by the highest occupied molecular (HOMO) where Avi(r) is the external field that molecule i experiences density divided by two (13). The geometry of the molecules because of molecule j [we ignore the Avi(r) caused by the were optimized at the self-consistent field/6-31** level. To control the conformation of the molecule, the FREEZE change in geometry of molecule i]. Similarly, Avj(r) is the external field that molecule experiences command was used in GAMESS. The dihedral angle between the j because of molecule carboxylate group and the C-C double bond was fixed during i. In Eq. 3, qi is the global hardness of molecule i and rj is the the optimization procedure of the calculation. All calculations global hardness of molecule j. To understand the meaning of were carried out on the Maui High-Performance Computer Eq. 3, one can write: Center IBM model SP2 high-performance parallel computer. All the calculations converged and the equilibrium geometry was checked and shown to have one constraint. /E2otal2 Jsi(r) vi(r)dr! RESULTS AND DISCUSSION The Conformational Modulation of the Reactivity of PEP. + + A PEP has both C=C and C=O double bonds; the relative 2j(f s(r)Avi(r)dr) [5] orientation of the two planes of these bonds may be used by an enzyme to modulate the reactivity of PEP. This is loosely where analogous to pyridoxal phosphate-dependent enzymes (14), in which the relative orientation of the oa bond and the XT plane of the conjugated system is believed to be the determinant for A = (gi - tj)A N + (rji + qj)AN2 + f[pi(r)Avi(r) the chemical course of the reaction. Thus, the C-H bond, C- C bond, or C-N bond can be cleaved depending on its orientation to the ir system, and is the only example of enzymatic reaction mechanisms in which rotameric conforma- + pj(r)Avj(r)]dr + ANJ [fi(r)Avi(r) tions can be controlled by a particular enzyme to influence the outcome of the reaction. To explore the possibility that a similar mechanism controls PEP reactivity, the energy, the a )2 charge, and the Fukui function of PEP of differing conforma- -fA (r)A vj(r)]dr 2f[si(r)(A vi(r))2 tions were examined by quantum chemistry methods. The results are listed in Table 1. Thirty conformations at three + sj (r)(Av1 (r))2]dr [6] different ionization states were studied in which the dihedral angle between the carboxylate plane and the enol plane was IfA is small when compared with the first two terms of Eq. 5, varied from 00 to 90°. In Fig. 2, the energy of PEP at different then the reaction energy is lowered if the local softness of conformations and different ionization states are plotted. For molecular i and j are minimized. In other words, hard likes each ionization state, the energy at 900 is used as the reference hard. Similarly, one can have Eq. 6: for that particular ionization state and the energy reported in the plot is relative to that. The energy needed to change the conformation of PEP is highest for the monoionization state, a t2 A Ettal= - J [si(r)(Avi(r))2 about 6.5 kcal/mol. For PEP-3, the single bond is almost freely rotating. It is clear that the conformational change of PEP is not very demanding energetically. At the active site of enzyme, + sj(r)(Avj(r))2]dr + B [7] one can imagine that the carboxylate group of PEP rotates around the single bond with little or no assistance from the where binding energy provided by the protein active site. Therefore, the role of the enzyme is to select one particular conformation B = - + + + (ii tj)A N (,qi -j) AN2 f[Pi(r)A vi (r) of PEP during catalysis; this selection has no particular ener- getic cost. On the other hand, the local properties of the molecule, such as the charge and the HOMO density, change + pj(r)Avj(r)]dr + ANJV(r)Avi(r) dramatically when the conformation of the molecule varies. For PEP-' and PEP-3, the total negative charge at C3 assumes a maximum when the dihedral angle between the two planes is close to 900 and assumes a minimum when the dihedral angle a - fh (r)Avj (r)]dr + ( fsi(r)Avi(r)dr) is close to 00 (Fig. 3A). The variation is linear. PEP-2 behaves more differently than PEP-' and PEP-3. Although the charge of C3 assumes a maximum at a dihedral angle of 70°, the charge drops rapidly when the dihedral angle changes from 700 to 600. + -ja(fsj(r) Avj(r)dr) [8] From 600 to 10°, the charge of PEP-2 decreases smoothly and slowly. At 00, the charge increases dramatically. Overall, the Downloaded by guest on September 26, 2021 4614 Biochemistry: Li and Evans Proc. Natl. Acad. Sci. USA 93 (1996)

coo- COO-

OH 0 OH 0P032-

'-03P 1. O OH 2-03PO OH OH OH OH OH

KD08P OH I 2-03P 0 II HO H HO Ara5P coo- 2-o3PO 0 3 0 HO"' "1H OH AH PEP O- XI4%%~II DAHP 2-o3PO N *4,., HO" %0 OH EPP 6H S3P COO0 I

2-03PO' .oJ coo OH EPSP FIG. 1. Schematic representation of three enzymatic reactions that use the enolpyruvyl moiety of PEP. The arrow in the structure of PEP indicates the rotation of the carboxylate group of the molecule around the single bond. Structures in parentheses indicate intermediates observed experimentally; structures in brackets indicate intermediates proposed here that are not yet observed. Path I is the 5-enolpyruvyl-shikimate-3- phosphate (EPSP) synthase catalyzed reaction; path II is that 3-deoxy-D-manno-2-octulosonate-8-phosphate (KD08P) synthase catalyzed reaction; and path III is the 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzed reaction. variation of the charge with dihedral angle is not linear but ingful parts of the electronic structures of PEP. The message parabolic with a minimum charge at about 300. At this angle, is very clear: the reactivity of PEP is modulated at a funda- a reaction involving PEP, which is determined by charge, has mental level by controlling the charge, the HOMO density, and the lowest reactivity. In contrast, PEP-' and PEP-3 have the the ionization state. lowest charge at 00. All three ionization states have the highest Implications In Enzymatic Catalysis. The reaction catalyzed charge at C3 when the dihedral angle is close to 900, and thus by EPSP synthase. EPSP synthase catalyzed forward reaction is at this conformation a charge-determined reaction will have optimal from pH 6 to 7.5 (17). At this pH range, when bound the highest reactivity. The general trend in the changes of the to the enzyme, PEP may exist in the PEP-2 form (18), HOMO density at C3 of PEP is about the same as that of assuming the phosphate pKa is perturbed by -0.5 pH unit. The charge except for PEP-2 (Fig. 3B). For a soft reaction, the observed deuterium kinetic isotope effect for the forward conformation closest to 900 is clearly the best choice (both for reaction implies that proton transfer is the rate-limiting step total charge and HOMO density). The energy required to (1). For a protonation reaction, the Fukui function (or HOMO reach this conformation is less than 3 kcal/mol for both PEP-2 density) of the reaction site should be minimized (11). This and PEP-3. At room temperature, this is a freely accessible occurs at C3 of PEP-2 when the dihedral angle is around 300. state. For PEP-1, because the carboxylate group is protonated Therefore, this conformation might be selected by EPSP and if this is hydrogen bonded to an active-site amino acid synthase for the reaction. This hypothesis can be tested by side-chain, the energy required to access this conformation time-resolved solid-state NMR spectroscopy in which the may be compensated for. However, for a hard reaction, a site transient species of the PEP-enzyme complex is to be trapped of minimal HOMO density is the most reactive. For PEP-' and on a fast timescale; these experiments are underway in this PEP-3, this occurs when the dihedral angle is 00, whereas for laboratory (19, 20). The enzymatic mechanism for EPSP PEP-2 this occurs when the dihedral angle is around 300. Thus, synthase catalyzed reactions may be described as follows. A the theoretical calculations at the self-consistent field/6-31** proton is transferred to C3 of PEP from an active site residue level reveal two distinct conformations that may be relevant to to initiate the enzymatic reaction. The cationic transition state enzymatic catalysis and that could be examined experimentally generated is highly reactive toward the 5-hydroxyl of shiki- by using solid-state NMR methods (15, 16). mate-3-phosphate, which sits close to PEP at the active site of Although the quantum chemistry methods we have used do EPSP synthase (we have docked PEP and shikimate-3- not include solvation effects and the protein microenviron- phosphate to the active site of EPSP synthase based on the ment, to a first approximation, they reveal the more mean- enzyme crystal structure and found that both substrates would Downloaded by guest on September 26, 2021 Biochemistry: Li and Evans Proc. Natl. Acad. Sci. USA 93 (1996) 4615

Table 1. Self-consistent field/6-31 * * calculations of energy, net charges (q), and the Fukui functions (f) of PEP at different ionization states and dihedral angles Molecules Angles Energy x 103 q(3) f(3)- 0 3 0.0 -568.51631 -0.3313 0.4053 10.0 -568.51675 -0.3356 0.4143 20.0 -568.51700 -0.3432 0.4350 11- ,OH 30.0 -568.51681 -0.3520 0.4575 40.0 -568.51300 -0.3492 0.4461 HO O' 50.0 -568.51187 -0.3531 0.4617 60.0 -568.51294 -0.3864 0.4652 70.0 -568.51200 -0.3918 0.4765 80.0 -568.51112 -0.3948 0.4839 PEP-1 90.0 -568.51050 -0.3955 0.4910 0 3 0.0 -568.08625 -0.4459 0.4823 10.0 -568.08306 -0.3918 0.4325 2 0~~~o 30.020.0 -568.08262-568.08294 -0.4037-0.3976 0.41320.4172 HO o 40.0 -568.08212 -0.4109 0.4175 50.0 -568.08144 -0.4183 0.4277 60.0 -568.08056 -0.4250 0.4434 O 70.0 -568.08388 -0.4779 0.4984 80.0 -568.08363 -0.4804 0.4809 PEP-2 90.0 -568.08363 -0.4809 0.4927 O 3 0.0 -567.54194 -0.4337 0.4974 10.0 -567.54194 -0.4341 0.4962 20.0 -567.54194 -0.4356 0.4950 011'u'.p2 0~~~~o 30.0 -567.54219 -0.4488 0.5235 -0 0 40.0 -567.54219 -0.4555 0.5346 /O 50.0 -567.54188 -0.4620 0.5469 60.0 -567.54169 -0.4666 0.5581 70.0 -567.54137 -0.4695 0.5678 80.0 -567.54106 -0.4707 0.5739 PEP-3 90.0 -567.54088 -0.4697 0.5744 PEP-n stands for PEP in different ionization state (n is the charge number). Angles refer to the dihedral angles between the carboxylate plane and the enol plane, in degrees. Energy is in kcal/mol.

appear to bind to the same domain of the enzyme and sit next face addition of PEP to the re face of the carbonyl of the to each other). As a result, a tetrahedral intermediate is cosubstrate erythrose-4-phosphate implies that C3 is the initial formed, which has been detected both at the active site of the reaction site in PEP (30). The pH optimum for DAHP synthase enzyme (20-22) and in the isolated chemical state (23). is 6.8 (31), which is much less than the pH 9 required for The reaction catalyzed by KD08P synthase. In contrast to the KD08P synthase. PEP exists in the PEP2- form in solution and EPSP synthase-catalyzed reaction, in the KD08P synthase at the active site of the enzyme. Again, for the reaction occur catalyzed reaction a soft basic atom (an atom with large rapidly, the dihedral angle between the C=O and C=C HOMO density) is required at C3. This can occur for all three double bonds should be close to 70° (although the HOMO ionization states of PEP when the dihedral angle is about 900. density at 00 is about the same as that at 700, the charge favors However, the HOMO density of PEP-3 is significantly higher the 700 conformation). In both the DAHP synthase and the than that of PEP-' and PEP-2, so that one would predict that this is the ionization state that would be favored for enzymatic 4.00 catalysis. One of the pH optima for the KD08P synthase catalyzed reaction is -9 (24); at this pH, PEP-3 is the most stable form in solution. When PEP binds to the enzyme, its co 2.00

phosphate group may be hydrogen bonded to an active-site 0)a) amino acid residue, but PEP probably will still exist in the 0.00 PEP-3 form. group not Clearly, the carboxylate should be L- hydrogen bonded to the enzyme because this decreases the a) -2.00 HOMO at C3 of PEP. The observation that the density cs enzymatic reaction is stereospecific, with si face addition of -4.00 PEP to the re face of the of Cl) carbonyl D-arabinose-5-phosphate, co another substrate of the enzyme, implies that the C3 is involved -6.00 in the rate-limiting step (25, 26). This is supporting evidence cn that the local property at C3 determines the selectivity of the reaction. -8.00 0 20 40 60 80 The reaction catalyzed by DAHP synthase. As in the KD08P Dihedral angles synthase catalyzed reaction, a soft base at C3 is needed for the reaction catalyzed by DAHP synthase to attack the carbonyl FIG. 2. Plot of the energy against the C1-C2 dihedral angles of PEP carbon (27-29) (Fig 1, path III). Indeed the stereospecific si for different ionization states. 0, PEP-'; 0, PEP-2; A, PEP3. Downloaded by guest on September 26, 2021 4616 Biochemistry: Li and Evans Proc. Natl. Acad. Sci. USA 93 (1996) the Pareto principle, efficiency is when partners are A highest both well-satisfied. For an enzymatic reaction, the same prin- -0.300 ciple can apply. An enzyme can select a specific substrate 0, conformation at its active site; the correct conformation thus

0- produces the correct charge and Fukui function for a particular o -0.350 desired reaction. Because of the match of the local softness and charge between two interacting substrates or between sub- 0CZ strate and enzyme, the total activation energy of the reaction is reduced. Therefore, the HSAB principle also provides an a) -0.400 added insight into Pauling's transition-state stabilization the- 0) ory proposed almost 50 years ago (33). We acknowledge the Maui High-Performance Computer Center for a) -0.450 access to and use of the IBM model SP2 parallel computer for the calculations reported here. This work was supported by the National Institutes of Health (GM43215). -O.500 1. Grimshaw, C. E., Sogo, S. G., Copley, S. D. & Knowles, J. R. 0 20 40 60 80 Dihedral (1984) J. Am. Chem. Soc. 106, 2699-2700. angles 2. Anton, D. L., Hedstrom, L., Fish, S. M. & Abeles, R. S. (1983) B Biochemistry 22, 5903-5908. 3. Unger, F. M. (1981) Adv. Carbohydr. Chenm. Biochem. 38, 323- 0.600 388. 4. Hedstrom, L. & Abeles, R. (1988) Biochem. Biophys. Res. Com- CL LU mun. 157, 816-820. 0~ 5. Pearson, R. G. (1963) J. Anm. Chem. Soc. 85, 3533-3539. 0.550 6. Pearson, R. G. (1966) Science 151, 172-177. 7. Parr, R. G. & Pearson, R. G. (1983) J. Am. Chem. Soc. 105, C1) 7512-7516. 8. P. K., Lee, H. & Parr, R. G. (1991) J. Am. Chenm. Soc. 0.500 Chattaraj, a) 113, 1855-1856. 9. Parr, R. G. & Yang, W. (1984)J. Am. Chem. Soc. 106,4049-4050. C0 10. Fukui, K. (1971) Acc. Chem. Res. 4, 57-64. 0 -o 0.450 11. Li, Y. & Evans, J. N. S. (1995)J. Am. Chem. Soc. 117, 7756-7759. 0 12. Schmidt, M. W., Baldridge, K. K., Boats, J. A., Elbert, S. T., I7 Gordon, M. S., Jensen, J. H., Koseki, S., Matsunaga, N., Nguyen, K. A., Su, S. J., Windus, T. L., Dupuis, M. & Montgometry, J. A. 0.400 (1993) J. Comput. Chem. 14, 1347-1363. 0 20 40 60 80 13. Yang, W., Parr, R. G. & Pucci, R. (1984)J. Chem. Phys. 81, 2862. Dihedral angles 14. Dunathan, H. C. (1966) Proc. Natl. Acad. Sci. USA 55, 712-716. 15. Evans, J. N. S. (1995) Biomolecitlar NMR Spectroscopy (Oxford FIG. 3. Plots of the effects of C1-C2 dihedral angles of PEP on Jniv. Press, Oxford). charge (A) and HOMO density (B) for different ionization states. 0, 16. Tomita, Y., O'Connor, E. J. & McDermott, A. E. (1994) J. Am. PEP- I; *, PEP-2; A, PEP-3. It can be seen that the charge is Chem. Soc. 116, 8766-8771. modulated by the conformational change. C3 of PEP can be a soft base 17. Shuttleworth, W. A. & Evans, J. N. S. (1994) Biochemistry 33, or a hard base, depending on the conformation. 6812-6821. 18. Wold, F. & Ballou, C. E. (1957) J. Biol. Chem. 227, 301-328. KD08P synthase catalyzed reactions, because C3 of PEP is the 19. Evans, J. N. S. (1995) Encyclopedia of NMR (Wiley, New York), reactive site and acting as a soft base (large HOMO density), pp. 4757-4763. at C3 of is minimized 20. Appleyard, R. J., Shuttleworth, W. A. & Evans, J. N. S. (1994) protonation (e.g., hydrolysis PEP) during Biochemistry 33, 7062-7068. the catalysis because the proton prefer to attack a site of small 21. Barlow, P. N., Appleyard, R. J. & Evans, J. N. S. (1989) HOMO density. This highlights the importance of the HSAB Biochemistry 28, 7985-7991, and erratum (1989) 10093. principle in enzymatic catalysis; without making use of it, an 22. Evans, J. N. S., Appleyard, R. J. & Shuttleworth, W. A. (1992) J. enzymatic reaction would not be specific (giving rise to side Am. Chem. Soc. 115, 1588-1590. reactions such as hydrolysis) or efficient. 23. Anderson, K. S., Sikorski, J. A., Benesi, A. J. & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579. CONCLUSION 24. Ray, P. H. (1980) J. Bacteriol. 141, 635-644. In conclusion, quantitative calculations on the Fukui functions 25. Kohen, A., Berkovich, R., Belakhov, V. & Baasov, T. (1993) and the of PEP have shown that the Bioorg. Med. Chem. Lett. 3, 1577-1582. charges regioselectivity of 26. Dotson, G. D., Nanjappan, P., Reily, M. D. & Woodard, R. W. PEP is controlled by its ionization state and conformation. (1993) Biochemistry 32, 12392-12397. This reveals a new family of enzymatic reactions, besides 27. Srinivasan, P. R. & Sprinson, D. B. (1959) J. Biol. Chenm. 234, pyridoxal phosphate-dependent enzymes in which the reac- 716-722. tions are modulated through the change in the conformation 28. DeLeo, A. B. & Sprinson, D. B. (1968) Biochem. Biophys. Res. of the substrate. The proposed mechanism of conformationally Commun. 32, 873-876. modulated reactivity can be extended to other PEP-using 29. Floss, H. G., Onderka, D. K. & Carroll, M. (1972)J. Biol. Chem. enzymes. Furthermore, the modulation mechanism has been 247, 736-744. found to be related to the intrinsic of the substrate. 30. Pilch, P. F. & Somerville, R. L. (1976) Biochemistry 15, 5315- properties 5320. Thus, the enzymatic mechanisms involving PEP can be un- 31. Schoner, R. & Herrmann, K. M. (1976) J. Biol. Chem. 251, derstood in terms of the HSAB principle. 5440-5446. In the elegant paper by Chattaraj and colleagues (8), the 32. Samuelson, P. A. & Nordhaus, W. D. (1989) Econonmics analogy was drawn between the Pareto principle (32) in (McGraw-Hill, New York), 13th Ed., pp 747-748. economics and the HSAB principle in chemistry. According to 33. Pauling, L. (1946) Chem. Eng. News 24, 1375. Downloaded by guest on September 26, 2021