Identifying sequential substrate binding at the single-molecule level by enzyme mechanical stabilization.

ABSTRACT: Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single-molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic adenine diphosphate (ADP)-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate 1, which is stabilized in the presence of Mg·ADP(-), the first substrate to bind to the enzyme. However, in the presence of this substrate, an additional unfolding event is observed, intermediate 1*. Finally, in the presence of both substrates, the unfolding force of intermediates 1

SUBMITTER: Rivas-Pardo JA 

PROVIDER: S-EPMC4467879 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature