The allosteric impact of the variable insert loop in Vaccinia H1-related (VHR) phosphatase.

Dynamics and conformational motions are important to the activity of enzymes, including protein tyrosine phosphatases. These motions often extend to regions outside the active site, so-called allosteric regions. In the tyrosine phosphatase, Vaccinia H1-Related (VHR) enzyme we demonstrate the importance of the allosteric interaction between the variable insert region and the active site loops in VHR. These studies include solution NMR, computation, steady-state, and rapid kinetics measurements. Overall, the data indicate concerted millisecond motions exist between the variable insert and catalytic acid loop in WT VHR. 150 ns computations studies show a flexible acid loop in WT VHR that opens during the simulation from its initial closed structure. Mutation of the variable insert residue, asparagine 74 to alanine results in a rigidification of the acid loop as observed by molecular dynamics simula-tion and a disruption of crucial active site hydrogen bonds. Moreover, enzyme kinetics analysis shows a weakening of substrate affinity in the N74A mutant and an over 2-fold decrease in substrate cleavage and hydrolysis rates. These data show that despite being nearly 20Å from the active site, the variable insert region is linked to the acid loop by coupled millisecond motions and that disruption of the communication between the Variable Insert and active site alters the normal catalytic function of VHR as well as perturbing the active site environment.