SDS-PAGE analysis of TM0436. Elution 1 displays purified protein with a clear band around 40 kDa
Research Design & Methods
The further study of TM0436 function will require more exact kinetic data, and most importantly, the precise identification of the unknown ligands. There is a wide range of suggested substrates which need to be examined against the protein for ligand binding and activity profiles. More useful information could be gained from identifying other cofactors and enzyme inhibiting molecules as well. In order to address these questions, an electrochemistry approach will be followed, due to TMO436’s oxidoreductase activity. Electrochemical methods have been widely used for the direct studies of electron transfer reaction kinetics and thermodynamics in proteins [8]. One way in which studies of this
nature can be carried out is through the use of amperometric enzyme electrodes, which couple the redox catalysis of the enzyme to electron transfer at the electrode surface. Enzyme electrodes can provide high specificity and sensitivity for inquiry into enzyme kinetics and ligand binding.
Carbon-fiber Microelectrode (CFME).
In this study, the purified recombinant protein will be immobilized on a carbon fiber microelectode (CFME) surface to investigate protein-ligand binding and enzyme kinetics. 3 µm-radius CFMEs are made using T-65O carbon fiber which is drawn through a glass capillary tube by vacuum aspiration. The CFME will be used as the working electrode in the electrochemistry apparatus. An Ag/AgCl wire will be used as the reference electrode for all experiments. Data will be acquired using Tarheel CV software and a GeneClamp potentiostat.
Fast-Scan Cyclic Voltammetry.
Ligand-binding experiments will involve exposing the electrode surface to various concentrations of possible subtrates, including a variety of short-chain alcohols as well as threonine. Current spikes will indicate binding of a ligand, and the time response and amplitude of the signal will provide details as to which ligands have higher specificity for the enzyme binding site. Kinetics will be examined at substrate concentrations within, above and below the Michaelis-Menten constant (Km) value for the enzyme. Maximum reaction velocity (Vmax) and Km values will be compared with previously computed values from other methods compared, and the effects on electrode response time and reaction kinetics by possible inhibitors will be explored. A pH profile of the enzyme will be obtained and used to construct a calibration curve. Apparent Km and Vmax values for different substrates will be determined using double-reciprocal plots. Lifetimes of the constructed enzyme electrodes will also be determined. For each set of measurements, at least three electrodes will be used to ensure reproduciblity and avoid electrode bias.