Overview

ThDPEnzymology can mean different things to different people, and bioorganic chemistry has an even broader scope. In our laboratory we consider enzymes in the context of their homologues: other proteins that have evolved from a common precursor. This approach seeks to identify common strategies for classes of enzymes toward catalyzing classes of reactions. By understanding more about one enzyme, we thereby learn about many enzymes.

Every individual project carried out in the lab uses a combination of techniques to address the research questions. Members of the Palmer Lab have the opportunity to learn some or all of the techniques described below.

Synthesis of substrates, pseudo-substrates, and inhibitors

Enzymes are catalysts, and studying the reactions they catalyze is the primary way to understand them. Some enzymes act on substrates that are cheap and available, but many do not, and as chemists we use synthetic organic chemistry to prepare the required substrates. One can develop a hypothesis regarding how the enzymatic reaction occurs, and test that hypothesis by using synthetic substrate analogues. The analogues may also undergo reaction or they may inhibit or inactivate the enzyme. Rational design of such compounds can allow us to support or disprove a proposed enzyme mechanism.

Effective inhibitors may be candidates for novel drugs. Such candidates can be tested on target organisms directly to assess their effects. We are also interested in developing compounds that can be used as novel medical imaging agents.

Molecular biology and protein chemistry

Our lab typically works with recombinant enzymes, meaning we isolate and maintain ("clone") the DNA encoding the protein sequence into an appropriate expression vestor, and express the gene in E. coli. The protein is isolated from the bacterial culture and purified by chromatography. In some cases, we will clone biosynthetic pathways so that we can learn about the function of a set of enzymes working together. If we have some understanding of how an enzyme works, we test our hypotheses by site-directed mutagenesis, the variation of a selected residue or residues, and observe the results on the catalyzed reaction.

Enzyme kinetics and biophysical characterization

The dependence of the rate of the catalyzed reaction on substrate, cofactor, an/or inhibitor concentration can yield information regarding the enzyme mechanism. Kinetic isotope effects can identify rate-limiting steps, and in some cases help us describe the transition state of the reaction.

Biophysical experiments gives alternative means of understanding proteins. For example, isothermal titration microcalorimetry allows measurement of thermodynamics of protein-ligand binding, even if no reaction is taking place. The facilities of the Saskatchewan Structural Sciences Centre (SSSC) allow us to use several such techniques in our work.

Protein crystallography is an important tool in studying enzymes, but this is not a crystallography lab. We collaborate with other laboratories in order to generate high-resolution structural information.

Computational approaches

Sequence comparison is a key tool used to predict protein function and determine phylogenetic relationships. We are not bioinformaticians, but we use these tools in order to examine structure-function relationships. In collaboration with Dr. David Sanders, we also use molecular modeling of both proteins and substrates as a means of predicting or rationalizing experimental results.

Please see the publications page to learn more about how we apply these techniques.


Are you interested in joining the Palmer Lab?
 

If you are an undergraduate looking for a research project for your degree program, or in a summer research position, please send an e-mail.
If you are interested in graduate studies, please send an e-mail after reading the instructions found here.
Applications for postdoctoral research positions are welcome from applicants eligible for external funding programs.