Protein Dynamics in Catalysis by LDH and DHFR

Project: Research project

Project Details

Description

The central aim of this study is to understand the dynamical nature of hydride transfer in the family of enzymes that use NAD/NADP as cofactors with lactate dehydrogenase (LDH) and dihydrofolate reductase (DHFR) as model systems. It is known that dynamical features of these two proteins are important for function. The two proteins are important model systems for comparison since LDH is a more 'rigid' protein compared to (E. coli) DHFR and for experimental reasons. The approach for our study is T-jump relaxation spectroscopy employing UV/vis absorption and fluorescence emission and mid-IR absorption to follow changes in structure from ps to minutes (or longer), some 15 decades of time. These probes provide substantial structural specificity, and preliminary studies exhibit dynamical features never before observed on any enzymic system. The studies are designed to probe the kinetics and structural changes of substrate-product inner conversion of on-enzyme chemistry over the entire ps-minutes time range and characterize fast hydride and proton transfer steps, loop motion, motions that modulate electrostatic catalysis, relative atomic motion between the bound substrate and active site residues, and the effects on the structure and dynamics at the active site by protein motions far from the active site. The role of interconversion kinetics of protein sub-states between catalytically active and inactive protein sub-states and 'promoting vibrations' on on-enzyme catalysis are tested. We shall probe and characterize the dynamics of protein complexes that mimic Michaelis complexes, complexes that mimic some aspects of the transition state, as well as productive enzyme/substrate enzyme/product inner conversion. In addition, the effects of key protein mutants on the dynamics will be studied in order to relate dynamics to catalysis.
StatusFinished
Effective start/end date5/1/044/30/09

Funding

  • National Institute of General Medical Sciences: $267,017.00
  • National Institute of General Medical Sciences: $160,869.00
  • National Institute of General Medical Sciences: $266,636.00
  • National Institute of General Medical Sciences: $164,265.00
  • National Institute of General Medical Sciences: $264,849.00

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