Essentially, an enzyme assay is taking an enzyme and combining it with the starting materials. Therefore, it is necessary to have a way to monitor the disappearance of the starting materials or the formation of the products. If the enzyme does increase the rate of the reaction, then the formation of products will be noticeably faster with the enzyme than without the addition of the enzyme. Because the catalyst being used here comes from a living organism, it is sensitive to salt concentration, pH, and the concentrations of the starting materials and products. Literature on the protein can give useful information on these values.
When beginning the initial crude assays, do not stress out – the likelihood of it looking correct on the first try is extremely slim. There are too many factors that will need to be tweaked to get good data. When starting out, give the assay the best chance of showing catalytic activity, without wasting time being precise with measurements and making stock solutions.
Deep Thoughts on Kinetics and Assays
You will be at least comparing the kinetics of your native protein to ncAA-mutant protein using an assay. But what do these changes in kinetic parameters actually mean and how do you interpret your results? Interpreting your results in a meaningful way may be one of the most challenging parts of your experiments.
First, let’s brush up on some basic kinetics values. KM is the Michaelis constant and is the substrate concentration at 1/2 Vmax. Vmax is the maximum velocity of the enzyme. kcat is turnover number and measures the number of substrate molecules turned over per enzyme molecule per second. kcat/KM is often used as a measure of catalytic efficiency. How are you going to obtain these values (and perhaps others) using your kinetic assay?
Typically we assume enzymes follow Michaelis-Menton kinetics, but is this true for your enzyme? What is the most appropriate model with which to model the kinetics of your enzyme and how has it been modeled in the literature? To assess the kinetics of your proteins, at what time points should you take your measurements and for how long? What other factors do you need to consider in order to conduct an accurate and consistent kinetic assay? For example, how do temperature, pH, and buffer composition affect your assay? What variables should be constant and which variables should change as your carry out your assay? (If your enzyme follows Michaelis-Menton kinetics, enzyme concentration should stay the same and substrate concentration should change.)
Once you have your results, what do you do with them? How might you assess the accuracy and validity of your measurements? Are your results self-consistent and do they make sense? You will also be able to find accepted kinetic parameters for your native protein in literature. What makes for a meaningful comparison between your values and those found in literature? What do changes in the various kinetic parameters mean?
- Materials will vary by group depending upon the nature of the assay. Each group is responsible for ensuring that the necessary equipment and materials are available from week to week for executing their assays.
Suggested Resources and Protocols
- Assay Background Information: Chapter One of the book Enzyme Assays by Eisenthal and Danson covers many of the basics for running assays and performing kinetic measurements. A copy will be in the teaching lab.