COMPUTATIONAL STUDIES OF THE KINETIC ISOTOPE EFFECT INMETHYLAMINE DEHYDROGENASE

Abstract

There is currently experimental evidence of hydrogen tunnelling in over 20 different enzymes include yeast alcohol dehydrogenase (YADH), morphinone reductase (MR) and methylamine dehydrogenase (MADH). Various models have been used to describe hydrogen tunnelling in enzymes including the static barrier model, the vibrationally enhanced ground state tunnelling model (VEGST) and the environmentally coupled tunnelling model (ECT). Despite some differences in these models, there is a general consensus that a temperature dependent kinetic isotope effect (KIE) is indicative of tunnelling dominated by a ratepromoting motion.Stopped flow studies of MADH with ethanolamine as substrate (mm-MADH/EA) show that the KIE of the proton transfer decreases with temperature - within the framework of the ECT model, the kinetics of this proton transfer are consistent with ground state tunnelling dominated by active dynamics (a promoting vibration). However, an alternative hypothesis is that this temperature dependence can be attributed to the population of multiple reactive configurations within the active site. If distinct substrate configurations are associated with distinct kinetic behaviour, the temperature dependence of the KIE could be due to temperature dependent fluctuations in the relative populations of these configurations.Long and short time molecular dynamics simulations of mm-MADH/EA were carried out to explore both of these scenarios. Theethanoliminoquinone intermediate was found to adopt a number of different hydrogen bonding configurations in the active site of MADH. Adiabatic scans of the proton transfer event in conjunction with WKB calculations of the KIE showed that these hydrogen bonding patterns are associated with different barrier heights and KIEs. However, simple modelling with the Boltzmann distribution showed that fluctuations in the relative population of these configurations of the magnitude expected in the temperature range 278K-308K leads to negligible changes in the magnitude of the KIE. This suggested thatmultiple reactive configurations are unlikely to account for the temperature dependence of the KIE. Spectral density analysis of the short-time MD simulations was then carried out try to identify any promoting motions in mm-MADH/EA. Since no evidence of promoting motions was found, the origin of the temperature dependence on the KIE remains an open question: the analysis in this study was restricted to one of four possible proton transfers in this substrate (HI3-OD1). Further work might look at the possibility of a promoting motion pertinent to the other transfers.

Date of Award	1 Aug 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Sutcliffe (Supervisor)