UCSF

Analyzing Enzyme Conformational Dynamics, Substrate Binding, and Catalysis

There are four research areas in the Department of Pharmaceutical Chemistry. Analyzing enzyme conformational dynamics, substrate binding, and catalysis is a research challenge within chemical biology and medicinal chemistry.

The challenge

Proteins routinely assume multiple shapes over time. The approximately 15 types of cytochrome P450 (CYP) enzymes in the human liver that metabolize most drugs are an important and extreme example of this shapeshifting, or conformational, dynamics.

The same versatility that allows them to bind and catalyze such a vast variety of compounds—reflected at the molecular level in their extra conformational flexibility and dynamics—makes it especially difficult to predict which substrates a given CYP isoform will act on and to what extent.

Since these CYPs oxidize xenobiotics to make them more water-soluble for excretion, thus activating or deactivating drugs and sometimes increasing toxicity, such information is vital in efficiently developing safe and effective drugs.

Examples of our research and methods include

Using unnatural amino acids to track molecular motion

Working with a model CYP from a bacterium, department researchers experimentally determined structures of the enzyme, both free and bound to a model ligand, using x-ray crystallography. They found dramatic conformational changes in the enzyme, as in human CYPs.

To determine how such molecular motion contributes to enzyme function, they expressed the bacterial CYP enzyme with unnatural amino acids at key locations to provide enhanced tracking of conformational dynamics via nuclear magnetic resonance (NMR) spectroscopy. Thus they were able to track molecular motions during binding events taking place over mere millionths of seconds.

Early findings suggest that instead of just adapting the shape of its active site to a given ligand (induced fit), the CYP enzyme normally cycles through various conformations and is shifted toward a form that cooperatively favors (or inhibits) catalysis via binding events at its allosteric sites.

Predicting specificity of human CYP isoforms

human cytochrome P450 2C9 enzyme binding warfarin

A human cytochrome P450 2C9 enzyme binding the blood-thinning drug warfarin. Sticks at center represent atoms of enzyme’s heme cofactor—gray carbon, blue nitrogen, red oxygen, orange iron—binding warfarin (at right)—gray carbons, red oxygen. Molecular image made with UCSF Chimera developed by UCSF Resource for Biocomputing, Visualization, and Informatics.

These research methods will be applied to human cytochrome P450 enzymes, which are more challenging to analyze because they are larger molecules and are membrane-bound in cellular organelles.

The ultimate goal is to combine these findings with computer models to construct protocols for predicting each CYP isoform’s affinity for specific substrates. One of the test models is CYP2C9, which metabolizes about 100 drugs, from warfarin (a commonly used blood thinner) to ibuprofen.