Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate by using NADH. LDH kinetics has been proposed to be dependent on the dynamics of a loop over the active site. Kramers' theory has been useful in the study of enzyme catalysis dependent on large structural dynamics. In this work, LDH kinetics was studied in the presence of trehalose and at different temperatures. In the absence of trehalose, temperature increase raised exponentially the LDH V-max and revealed a sigmoid transition of K-m toward a low-affinity state similar to protein unfolding. Notably, LDH V-max diminished when in the presence of trehalose, while pyruvate affinity increased and the temperature-mediated binding site transition was hindered. The effect of trehalose on k(cat) was viscosity dependent as described by Kramers' theory since V-max correlated inversely with the viscosity of the medium. As a result, activation energy (E-a) for pyruvate reduction was dramatically increased by trehalose presence. This work provides experimental evidence that the dynamics of a structural component in LDH is essential for catalysis, i.e., the closing of the loop on the active site. While the trehalose mediated-increased of pyruvate affinity is proposed to be due to the compaction and/or increase of structural order at the binding site.