Gating role of His 72 in TmPurL enzyme uncovered by structural analyses and molecular dynamics simulations.

Histidine is ubiquitous in enzyme active sites but its role is often difficult to ascribe due to ambiguity of protonation state and complex electrostatic and dynamic effects involved. In this study the role of His 72 in TmPurL, a glutamine amidotransferase (GAT) enzyme, is investigated. TmPurL is a large 66kDa enzyme that works as part of an even larger (>100kDa) multi-protein complex. This enzyme complex performs an essential step in the purine biosynthesis pathway by abstracting ammonia from a glutamine molecule and channeling it 30Å away into the active site of TmPurL, incorporating it into a purine biosynthesis intermediate. It is known that His 72 is important for substrate binding and possibly acts as a general base. Comparing apo and holo structural forms of this enzyme has revealed to us a possible gating function of His 72 that could regulate ammonia entry into the active site. Bimodal distribution of the χ dihedral angle of this amino acid in molecular dynamics simulations of 2μs supports the hypothesis. Different protonation states of His 72 were found to be conformationally distinct, providing a possible link between catalytic and gating roles of His 72. Ammonia channeling and allostery are discussed for GATs and more specifically for PurL family.