Investigating the Kinetic Effects of the Allosteric Interaction Between Newly‐Discovered Dynamic Hydrogen‐Bonded Residues and the HINT1 Active Site

Abstract

Histidine Triad Nucleotide binding proteins (HINTs) are an ancient class of enzyme notable for their acyl‐AMP hydrolase and phosphoramidase activity. This class of protein is of research significance due to its involvement in antiviral prodrug activation, as well as its natural role in opioid tolerance. Previously, the Wagner Lab has published the substrate specificity profile, full kinetic mechanism, and kinetics assay for the HINT isozyme HINT1. However, the active site dynamics of this enzyme have yet to be elucidated.Recent insights into the structural dynamics of HINT enzymes found via CPMG‐NMR and discovered by the Wagner Lab have revealed a potential site of regulation for the kinetic rates of HINT1. We hypothesize that a subtle allosteric interaction exists between the active site of HINT1 and the previously unrecognized distant site through a channel of conserved waters, which plays a key role in the active site dynamics and kinetics.Thus, with the goal of investigating the kinetic properties of this potential allosteric interaction, two genetic mutants of the enzyme HINT1 were produced to eliminate or alter the interactions between the highly dynamic residues Q62 and E100. Both steady‐state and stopped flow pre‐steady state kinetic analyses were performed on the mutants to determine any significant changes to their kinetics. Furthermore, circular dichroism analysis of both mutants was performed to ensure that both mutants were structurally sound, with no significant enzyme misfolding.Through these analyses, it was found that the kcat and Km of the HINT1 mutants were altered significantly and predictably through the pair of successive mutations. Through process of elimination, these findings informed us that these mutations to the dynamic residue Q62 must be altering the rate of intermediate hydrolysis at the HINT1 active site, a site previously thought unlikely to be altered.Support or Funding InformationPartial funding of this study by the University of Minnesota Office of Undergraduate Research is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.