Team:Washington/Gram Positive/Test

Enzyme Assay:

Reason:

Enzyme assay is commonly used to quantify the activity of an enzyme of interest by measuring the decrease in substrate or increase in produce concentration. Two main types: Continuous and discontinuous are the two main types of enzyme assay. While a continuation of measurement is made for a specified period of time, a discontinuous assay gives measurement for an instance of time. One advantage of a discontinuous assay is that it does not require one to make measurements of enzyme activity at time zero, but instead, one can wait to make measurements at the end of the reaction. However, making proper measurements, such as velocity, of an enzyme requires accurate measurement at the beginning of the reaction due to the varying in substrate and product concentration as a reaction progresses, thus, a continuation assay is used for the purpose of our project.

Medium of measurement:

Plate Reader

Under the category of continuous assay, various types of medium are measured for quantification. Spectrophotometric assay measures the change in the amount of light the assay solution consumes and this change is used to quantify the concentration of substrates or products. Calorimetric assay measures the amount of heat absorbed or released during a reaction to help detect the activity of an enzyme. For our project, fluorimetric assay is used to help measure the activity of enzyme CapDCP and its variants by detecting the change or difference in fluorescence emission of the substrate and product. (talk about detailed process) The greater the difference between the two emissions, the greater the enzyme activity occurred.

In measuring the difference between the two emissions, a plate reader is used. A plate reader illuminates the assay sample using a specific wavelength of light. The illumination triggers an emission of fluorescence and then a light detector measures the output emission.

Goal of project:

The goal of the project is to increase the hydrolysis capability of CapDCP. The desired result will be an increase in fluorescent detection for hydrolysis reaction. For this project, three conditions are assayed to determine whether a mutation contributes to an increase in hydrolysis reaction. The three conditions are: L-Glutamate, No L-Glutamate and a control that contains no enzyme. For the first condition, because L-Glutamate is the secondary substrate that assists in dissociating the covalent linkage of the primary substrate, PDG, and CapDCP, by binding to PDG and thus releasing and helping in regeneration of the CapDCP enzyme, we are testing the transpeptidation capability of the enzyme. PDG is translocated and re-attached to a secondary substrate, L-Glutamate. For the second condition, water is used in place of L-Glutamate as the secondary substrate used in releasing the enzyme from PDG. Water has somewhat lower affinity for our enzyme and therefore, is expected to result in a slower rate of regeneration and thus lower overall enzyme activity. The last condition contains all the ingredients except for the CapDCP enzyme.The ultimate goal for the project is to make mutant designs that will show an increase rate in the second condition, hydrolysis.

What we use for our project:

To assimilate the cleavage process occurred in in-vitro environment, we used a lab-safe PDG that contains a linked fluorophore and quencher. When the linkage is maintained, no emission will be expected from light illumination from the plate reader because the linkage transfers all the light energy received by fluorophore directly to the quencher. When all the exerted light energy is absorbed by the quencher through the transfer, no output light energy is exported for detection. When the PDG is cleaved by our enzyme, the embedded linked fluorophore and quencher is also disrupted. Illumination of light energy on the quencher and exported as fluorescent light and then can be measured by the light detector.

Data analysis and Purpose:

Before we can proceed on making mutations that can increase the hydrolysis capability, we need to validate that the circularly permeated version of CapD has measurable activity for further assessments. In order to validate our hypothesis that CapDCP has catalytic activity, we apply the same three conditions scheme to CapDCP as well as two knockouts. We also hypothesize that in the catalytic site of CapDCP, the tyrosine residue plays an important role in the catalysis reaction and mutating it will eliminate all enzymatic activities. The two mutants are T2V and T2A and act as controls. The result of this assay confirms our hypothesis that CapDCP has enzymatic activity relative to the two knockouts. The relatively flat activity curves of the knockout mutants also affirms the hypothesis on the important role of tyrosine in the catalytic site.

To validate the presence of our enzyme and be able to compare our circularly permeated CapD and the original CapD. Before we determine the kinectic properties of the two enzyme version, we decided to run a protein gel for the two to determine the physical traits on an argarose gel. Based on the gel, the two enzyme have apparently different band intensities with CapD having three bands and CapDCP having only one band. Two of the bands shown for CapD are equal in intensity but different in sizes. The bigger intensity band has the same size as the only band shown for CapDCP.

Reason we continue on with CapDCP:

Based on the result we see on the gel for the two enzymes, we decided to use CapDCP as the backbone for future mutant designs. The reason is two fold. First is that CapDCP is easier to quantify due to its single band characteristic shown on a gel. Second reason is that the three unequal and ambiguous band intensities can give us problems in the future since it is difficult to determine which band contains the active enzyme and thus very difficult to quantify our result. After we validated the activity of CapDCP, we proceeded on to making more analysis on the properties of the enzyme. We compiled a Michaelis-Menton profile to determine how we will modify our enzyme to achieve our goal. To determine the Michaelis-Menton profile, we ran a similar assay that employs our regular scheme. We ran the assay for both CapD and CapDCP enzymes. To determine the properties of the two enzymes, we ran the assay with varying substrate concentration so we can generate an activity curve for each enzyme. Some of the properties we are interested in are Vmax, Kcat, Km, Kcat/Km and Ki. Based on the Kcat and Km values of the two enzymes, we conclude that the properties of the two are very similar when taking in account of the uncertainties. Based on the gel result and the kinetic properties, we concluded that the two versions are very similar and all further designs will use CapD.

Analyzing the two abilities of CapDCP:

The two abilities of CapDCP are transpeptidation and hydrolysis. We apply the same assay scheme to determine the kinetic properties of the two reactions CapDCP can carry out. The Kcat and Km are two of the main factors we consider to assess the two abilities. Based on the Kcat and Km values of the two, we conclude that CapDCP is a weak binder and efficient catalyst for the transpeptidation reaction. For hydrolysis, it has shown strong binding but slow catalysis rate. Since our goal of the project is to increase the hydrolysis reaction, destroying the PDG completely instead of translocating it to another molecule, we focus on designing mutants to enhance the hydrolysis reaction of CapDCP. Since CapDCP is a strong binder in terms of its hydrolysis capability, we need to design CapDCP mutants that are strong binder and efficient catalyst in terms of hydrolysis ability.

Testing new designs:

To test whether our mutant designs meet our goal of enhancing the hydrolysis aspect of CapDCP, we again apply a similar assay scheme to each design. We standardize the activity slope of each design by dividing that of CapDCP to allow us make proper comparisons. Several of the designs have shown negative catalytic curves that are similar to the two knockouts. They are…Some have given intermediately negative activity curve that shows an decrease in either transpeptidation, hydrolysis alone, or in both. T20S is the mutant design that shows increase in hydrolysis activity.