Faculty Research Interest

Structural Determination of Paramagnetic Intermediates in Enzymatic Catalysis

Faculty Record

The goal of our research is to characterize protein function through the determination of intermediate state structures generated along a given reaction pathway. These intermediate states involve transient forms of the protein, cofactor and/or substrate. In a variety of enzyme systems, intermediates consist of paramagnetic species in the form of metals, organic radicals, or both metals and radicals. In addition, for systems which lack endogenous paramagnetic species, it is often advantageous to introduce a stable radical "spin label" to serve as a reporter of protein structure. Electron paramagnetic resonance (EPR) spectroscopy is well suited for the characterization of all of these classes of paramagnetic species. Thus our primary tools for structural characterization involve advanced EPR techniques, including electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR). A current focus in EPR development is the construction of a high frequency pulsed EPR/ENDOR spectrometer to extend the capabilities of EPR spectroscopy beyond magnetic field strengths available in commercial spectrometers.

Examples of systems currently under study include:

Glutamate Mutase: This enzyme catalyzes the reversible carbon skeletal rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate. EPR spectroscopy of radical intermediates is being used to unravel the mechanism of this rearrangement.

Ribonucleoside Triphosphate Reductase (RTPR): Because ribonucleotide reductases play a key role in DNA synthesis, their inhibition forms the basis of an important class of anti-tumor drugs. Both the turnover of substrate and the mechanism of inhibition involve organic radical intermediates, in addition to paramagnetic cobalt(II) present in the adenosylcobalamin cofactor. The study of RTPR function and inhibition is an ongoing research topic.