Source of catalysis in the lactate dehydrogenase system. Ground-state interactions in the enzyme-substrate complex.

The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which 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 frequency than observed for the carbonyl of unliganded pyruvate in the bacterial 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 enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C = O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C V O and the protonated His-195. Thus, while the C = O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stability substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)