The Role Of Conformational Collapse In Enzymic Catalysis

Lactate dehydrogenase catalyzes the inter-conversion between pyruvate and lactate, using NAD as cofactor. At the chemical step, hydride transfer from NADH to pyruvate C2=O carbon and proton transfer from protonated histidine to C2=O oxygen occur simultaneously on the ca. 20 fs time scale. Here, we investigate how (pig heart) lactate dehydrogenase (phLDH) guides the on-enzyme reaction pathway as the system goes from LDH⋅NADH⋅pyruvate to LDH⋅NAD+⋅lactate. Our previous studies on LDH⋅NADH⋅oxamate (a pyruvate analog) showed that this complex consists of two oxamate/NADH structures with different C2=O bond polarizations and different bending directions of the NADH ring. IR temperature-jump studies suggested these two structures do not interconvert directly but through a significantly less populated “encounter complex”. Here we report static FTIR and IR T-jump studies on LDH⋅NADH⋅pyruvate complex. This complex contains one major pyruvate structure in which pyruvate C2=O bond is significantly polarized. In addition, FTIR results can also identify at least three less populated pyruvate species with different C2=O bond polarizations. IR T-jump results suggest the pyruvate species with least C2=O bond polarization is likely the so called “encounter complex”, which is the gateway leading to the formation of other pyruvate species with more polarized C2=O bond in the ground state. We argue that the bulk of the rate enhancement of an enzyme, using the chemistry catalyzed by LDH as a model system, is best viewed as arising from a quantitative consideration of the energy landscape specific to that found in solution compared to that found in the enzyme-substrate system. The chemical event in either medium is at the tail end of a stochastic search, probably largely Markovian, through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational sub-states.