Part 1- Intro to Exploring Enzyme Activity: Research at Institute of Cancer Research at New York

Our research was centered around understanding how enzymes work at the molecular level, using a range of biochemical techniques.

Before we dive in, there is some background information that will be necessary to understand the experiment performed. To understand this experiment, it will require knowledge in the fields of biology and chemistry.

1) The Purpose of MDH2 in Cellular Metabolism

Malate dehydrogenase (MDH) is a key enzyme that plays a central role in cellular metabolism. It operates in the cytoplasm and other organelles, making it an intriguing model for studying enzyme activity and structure due to its varying properties across different cellular environments.

• Oxaloacetate: A crucial intermediate in the citric acid cycle, and reacts with acetyl-CoA to form citrate, continuing the metabolic process.

• Malate: Formed from fumarate, it is converted into oxaloacetate by MDH; prepares the system to process another acetyl-CoA molecule

2) Hydrogen bonding between MDH2 and NADH

The hydrogen bonding between MDH2 and NADH influences the enzyme's function. By mimicking serine phosphorylation through an aspartate mutation (due to structural similarities), we can keep MDH2 in a phosphorylated state, which is key to understanding its behavior.

3) Experimental Design:

Every experiment has a hypothesis or a question that is being solved.

Our question was: What is the effect of the S8D mutation on the rate of the forward reaction from oxaloacetate to malate? How does this compare to the wild type?

Hypothesis: Phosphorylation of S8 makes the interaction of NADH stronger and increases the rate of the forward reaction.

Some Limitations of the Experiment:

1. Only One Mutation Tested: We originally planned to test three mutations, but we only managed to successfully test the S8D mutation. This prevented us from seeing how the other mutations would have impacted the enzyme.

2. Small Sample Size: We only had a small sample size, testing just one colony of S8D mutated E. coli cells. This means we don’t know if the results would be the same if we had more colonies to test.

3. Fragile Competent Cells: The competent cells used during bacterial transformation were pretty fragile, so it was hard to grow colonies. This limited our ability to test all the mutations we wanted to.

4. No Control Group Colonies: Unfortunately, none of the colonies grew in our control group. Because of this, we had to rely on data from previous studies (a literature control), which may not have been as accurate for our exact conditions.

Our experiment involved introducing the mutation to the MDH2 gene through bacterial transformation. Here’s a glimpse of the process:

1. Bacterial Transformation

2. Protein Purification

3. Bradford Assay

4. Protein Gel

In the next post, we will go over the whole experiment in depth and the results/conclusion. See you there!