The major goal of this research project is to understand at the molecular level of biochemical and biophysical concepts underlying catalysis of p-hydroxyphenylacetate hydroxylase (HPAH), and pyranose oxidase. Each enzyme system will be investigated by various techniques and research methods including protein purification, kinetics, thermodynamics, and absorbance/ fluorescence/luminescence spectroscopy. Investigation on pre-steady state kinetics by using stopped-flow spectrometry will be one of our major tools to elucidate kinetic mechanisms. Specific aims for each system are described as below.
Specific Aims for the Study of p-hydroxyphenylacetate hydroxylase.
1. Investigate kinetics and thermodynamics of the oxygenase component (C2) by
using free FMNH2 or FADH2 as substrates. Kinetic mechanism of the reaction will
be elucidated and enzyme intermediates will be identified.
2. Investigate kinetics and thermodynamics of the oxygenase component (C2) by
using C1-FMNH2 as a substrate. Data will be compared to the reaction of free
FMNH2 in order to identify the effect of C1 protein on C2 reaction.
3. Propose the mechanism by which the FMNH2 is transferred between C1 and C2. We
will explore if our postulation is also applicable to other multi-component enzyme
systems where the transferring of the reactive intermediates exist.
4. Investigate kinetics and thermodynamics of the bacterial luciferase reaction by
using C1-FMNH2 as a substrate. Data will be compared to the reaction using free
FMNH2 in order to estimate an efficiency of this hybrid system.
Specific Aims for the Study of Pyranose Oxidase.
1. Investigate the reduction of the enzyme-bound FAD by various sugar substrates.
Rate constant associated with each step of the reaction will be compared among
various sugar reactions.
2. Investigate the oxidation of the reduced enzyme by oxygen and various electron
acceptors. The rate-limiting step of the reaction will be identified.
3. If possible, our study will suggest a better condition for using P2O to oxidize D-
galactose, a biotechnological process for synthesis of D-tagatose.
