An inquiry into the source of stereospecificity of lactate dehydrogenase using substrate analogues and molecular modeling.

Lactate dehydrogenase catalyzes the stereospecific hydride transfer to and from the re face of the nicotinamide coenzyme. The demonstrated probability of transfer to the si face of less than 2 x 10(-8) indicates that the free energy of any diastereotopic transition state leading to a si transfer must be over 10 kcal/mol greater than the free energy for transfer to or from the re face. The general notion of closed, desolvated active sites suggests the a priori hypothesis that steric hindrance prevents the nicotinamide ring from assuming a conformation that would lead to transfer of the pro-S hydrogen. In this paper we report that the probability of transfer of the pro-S proton is less than 9 x 10(-7) with 3-pyridinealdehyde adenine dinucleotide as coenzyme and less than 4 x 10(-7) during the lactate dehydrogenase catalyzed disproportionation of glyoxylate. Examination of the crystal structure of lactate dehydrogenase further suggests that steric exclusion does not enforce the extreme stereospecificity of the reaction. An electrostatic interaction with the macrodipole associated with the alpha 2F helix is suggested as a potential molecular source of the stereospecificity.