International Research Experience for Undergraduates

Jennifer Schefiliti

2002 Participant

Schefiliti

Oxalate Decarboxylase Enzyme

My research for this summer's work in Dr. Nigel Richard's group is centered around the oxalate decarboxylase enzyme. The goal of my research is to work to find a substrate for the enzyme, other than oxalate, and to be able to use this substrate to give information about the mechanism of the enzyme-substrate interaction. Additionally, an inhibitor will be selected and synthesised if the first two aims are completed.

To begin the research, I first synthesized several substrates. Benzyl ester oxalate, methyl ester oxalate and thio oxalate were the desired compounds. The thio oxalate has proven to be more difficult than previously expected and is in the process of being synthesized. The benzyl ester has been synthesized and purified and is the basis for next months work. The methyl ester has been synthesised as confirmed by both NMR and mass spec, but its purity has not yet been evaluated.

The work for the next month is centered around determining if the benzyl ester oxalate is a substrate for oxalate decarboxylase. A preliminary trial with impure material showed the compound to be a substrate. A significant amount of formate was produced in the assay. This months work will attempt to quantify previous results and to ensure that the interaction that is occurring is the expected one.

Several potential problems are being considered. First, oxalate was used as a starting material in the preparation of the benzyl ester oxalate. If even trace amount of oxalate were not removed in purification, the enzyme is so sensitive to oxalate that it will use the oxalate and produce formate. This is not expected to be the case because it would have to have been a 20% impurity, but this is being further investigated. Additionally, if the benzyl ester hydrolyzes in water or is hydrolyzed by the enzyme, it forms oxalate which could then be what is being used by the enzyme as a substrate. This too must be ruled out.

Current experiments are centered around resolving the afore mentioned complications and determining if benzyl ester oxalate is a substrate. If it is found to be a substrate, the methyl ester oxalate will be purified and experiments to determine its use as an inhibitor will be conducted.