276 2. ANALOGS OF ENZYME REACTION COMPONENTS 



tween hydroxyl groups and the Rl and R^ enzyme groups, polarization 

 of double bonds, and interactions of a third C00~ group (in the tricar- 

 boxylates), the tentative values shown in the following tabulation may be 

 assigned for the contributions made by the various interactions to the 

 total binding: 



Two C00~ groups 1.75 kcal/mole 



— OH group hydrogen bonding 0.5-1.5 kcal/mole 



— C=C — polarization 1.9 kcal/mole 



Additional CHgCOO- group 0.5 kcal/mole 



The hydrogen bonding and polarization values are minimal since, in part, 

 they were derived from the K„'s of the substrates (for fumarate K„^ = 

 = 1.78 mM and — JF = 3.71 kcal/mole, and for L-malate Z,„ == 4.0 mM 

 and — AF = 3.24 kcal/mole). The ^„/s may not represent dissociation 

 constants but in any case the true K^'s would be equivalent to or smaller 

 than the K„'s, so that the binding energies for the substrates may be 

 somewhat higher. The cis configuration of maleate reduces the attraction, 

 but the value for maleate in the table should be corrected since the pK^ 

 — 5.9 (at 23° and around 0.1 ionic strength) and only 74% of the total 

 maleic acid would be in the form of maleate=: this increases — JF to 2.82 

 kcal/mole. The difference in binding between fumarate and maleate is 

 thus at least 0.9 kcal/mole. It is also interesting that the introduction of 

 a methyl group into fumarate to form mesaconate brings about a 1.55 

 kcal/mole or greater reduction in the binding energy, resulting possibly 

 from a steric displacement and lowered polarization interaction. The 1.9 

 kcal/mole estimated for electrical polarization of the double bond is not 

 unreasonable and actually corresponds fairly closely to that calculated, 

 using appropriate molar refractions and an interaction distance of 4 A. 

 A factor of unknown importance is the possible deformation of the dicar- 

 boxylates to fit the active site and the energies that would be involved 

 with the different inhibitors. 



(B) If these conclusions are valid, the interaction energy for fumarate is 

 approximately half due to coulombic ion-ion forces and half due to the 

 inductive polarization by a strong dipole. It is possible that one of the R 

 groups on the enzyme is positively charged and the other negatively 

 charged on the active enzyme, as suggested by Massey (1953 b). The ionic 

 interactions serve to orient the fumarate at the active center, and the 

 polarization not only stabilizes the complex but initiates the addition of 

 water. 



(C) The third COO" group of citrate and trans-aconitate seems to be 

 able to interact with an adjacent positive group on the enzyme, but rela- 



