SOURCE OF CATALYSIS IN THE LACTATE-DEHYDROGENASE SYSTEM - GROUND-STATE INTERACTIONS IN THE ENZYME-SUBSTRATE COMPLEX

The Ramam spectra of both the NAd-pyruvate and the pyridine aldehyde a denine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, an d bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active8site cavity in these enzymes. The symm etrical stretching mode of the pyruvate carboxylate group, found at 13 98 cm(-1), is shifted only slightly when complexed to these enzymes, w hich shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm(-1) to a lower fre quency than observed for the carbonyl of unliganded pyruvate in the ba cterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C +-O- charge separation that was seen previously with the mammalian enz ymes. On the basis of an empirical Badger-Bauer relationship between f requency shift and interaction enthalpy, this shift in frequency is eq uivalent to an approximately -14 to -17 kcal/mol interaction between t he enzyme and the adduct C=O coordinate, a substantial part of which i s an electrostatic interaction (hydrogen bond) between the C=O and the protonated His-195. Thus, while the C=O bond is polarized on the enzy me (which requires energy), the overall ground-state enthalpy of the c arbonyl imidazolium part of the reaction coordinate is stabilized subs tantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other int ernal coordinates for the hydride transfer are not much affected durin g formation of the ternary complex. The total enthalpy of binding for pyruvate analogs to lactate dehydrogenase is nearly the same as the su m of local enthalpies for interactions between pyruvates C=O and the p rotein. Thus, even though ligand binding may cause any number of large , mostley compensating effects. The Raman spectra of the PAAD-pyruvate adduct bound to two different sets of mutant forms of the bacterial e nzyme also were measured. Mutation of Arg-109, which normally hydrogen bonds to the pyruvate C=O, to Gln-109, reduces the extent of C+-O- ch arge separation by about 4 kcal/mol. Similarly, mutation of Asp-168 or Ala-168 also reduces charge separation but to a somewhat greater degr ee, 4 and 10 kcal/mol, respectively. The ground state for the carbonyl imidazolium interactions of the mutant complexes is thus destabilized relative to the wild-type enzyme, and yet, the height of the transitio n-state barrier must increase, which clearly indicates the height of t he barrier must increase faster than the ground state is destabilized. This view is analyzed from a plot of the log of the hydride transfer step versus the change in frequency of the C=O stretch (or ground-stat e interaction enthalpy).