Characterizing Conformational Dynamics and Catalytic Activity of Enzymes by Hydrogen-Deuterium Exchange Mass Spectrometry

Determining the conformational dynamics of enzymes as they undergo catalysis has been challenging for decades due to the short timescale and limitations of traditional analysis, including techniques such as CPMG 2D NMR and X-ray crystallography. Herein is the study of two proteins: TEM-1 -lactamase, the most common enzyme responsible for the hydrolysis of antibiotics in gram-negative bacteria, and yeast alcohol dehydrogenase (YADH), responsible for facilitating the hydride transfer to NAD+ for energy production in prokaryotes. In the catalysis of both antibiotics and NAD+ reduction the specific residues involved in each binding mode remain under debate. By using a microfluidics workflow and an adjustable reaction volume, time resolved HDX experimentation was used to monitor deuteration events concurrently with catalytic activity on the millisecond to second timescale. Native mass spectrometry enabled studies into binding affinity and monitoring of substrate inactivation on a measured time course. Ion mobility mass spectrometry (IM-MS) was used to provide definitive MS/MS results for protein coverage and provide spatial resolution for all protein/substrate complexes. Additionally, collision induced unfolding (CIU) within the ion mobility cell provides a comparative binding affinity scale for the inhibitory drugs used in the study of TEM-1. Using this wide range of analytical techniques facilitated important discoveries including the isolation of specific residues of TEM-1 mapped to their involvement in different binding modes during catalysis, and subsequently the differentiation in inactivation pathways depending on substrate concentration and type. Further work has isolated residues involved in the turnover event of NAD+ along with residues that display a significant decrease in dynamics with the catalysis of deuterated ethanol compared to the non-deuterated ethanol.