Results

Phase 3: Characterization of Enzymatic Activity

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Background

The Ultraviolet–visible spectroscopy (UV-VIS) assay relies on the formation of 4 intermediates involved in the transamination of the substrate, each with a unique absorbance. This makes the reaction trackable using UV-vis spectroscopy, and by measuring the intensity of the absorbance peak, we can determine the activity of the reaction.

First, PLP covalently binds to the transaminase through an imine linkage with a lysine residue, forming a Schiff base. This structure is known as the internal aldimine, and it has an absorbance of 411 nm. An aspartate residue in the active site of the enzyme protonates the nitrogen in PLP’s pyridine ring, creating an electron sink for further reaction.

When the amine donor is added, it displaces the lysine residue to form the external aldimine, which has an absorbance of 330 nm. The external aldimine reacts with water to form the ketone byproduct and PMP, which has an absorbance at 314nm. The addition of the amine acceptor reforms the external aldimine, which then reacts to form the final amine product. The internal aldimine is regenerated, completing the catalytic cycle.

The covalent binding of PLP to the transaminase dimer is essential for its enzyme activity and to ensure that there was enough PLP to covalently bind to the purified transaminases, we incubated the enzymes with excess PLP before desalting it to remove excess PLP. This ensured that the UV-VIS spectrum wasn’t influenced by unbound PLP. Initial trials indicated that the transaminases worked best at concentrations close to 1 mg/mL. After concentrating HHTA and AATA, we performed the assay at 1 mg/mL of AATA and 0.8 mg/mL of HHTA (based on A280 absorbance). Both amine acceptor and amine donors were added to a final concentration of 2.5mM each. For AATA, L-phenylalanine was used as the amine donor while for HHTA, L-kynurenine was used as the amine donor. Due to a scarcity of previous studies on AATA, we tested two known amine acceptors that work on a variety of transaminases, sodium pyruvate and sodium phenylpyruvic acid as our control acceptors. HHTA is reported to accept sodium phenylpyruvic acid as an amine acceptor. For the production of 3,5-diiodo-L-tyrosine (“mini thyroxine”), our target product, we used the alpha keto acid of the compound, 3,5-diiodo-phenylpyruvic acid as our test amine acceptor.

Covalent binding of PLP to transaminases

As described above, when PLP covalently binds to the enzyme, it forms an internal aldimine ring. To detect this correct binding, we can measure the absorbance at 411 nm which corresponds to the formation of an internal aldimine. As seen in Figure 3, there is a distinct peak at 411 nm for both AATA and HHTA indicating the correct covalent bonding of PLP, as a cofactor, to the enzymes.

Enzymatic activity of HHTA

While we were successfully able to collect decipherable data on the enzymatic activity of AATA, our reading on the enzymatic activity of HHTA was skewed by the nature of the substrates required for the reaction in HHTA. L-kynurenine, the amine donor for HHTA, is a colored compound in solution and has significant absorption in the 300-600 nm range. It is also likely that our source of L-kynurenine had degraded because every reading of samples containing the substrate showed inconsistencies (See Supplementary Data). Due to this, we were unable to produce a reliable characterization of the enzymatic activity of HHTA. Moving forward, we would like to source different amine donors or re-try l-kynurenine with a more reliable methodology of activity assay, like mass-spectrometry.

Enzymatic activity of AATA with phenylpyruvic acid

Since we were unable to find reliable characterizations of transamination reactions with AATA we chose to try commonly accepted amine acceptors that are known substrates to multiple classes of transaminases. One such amine acceptor is phenylpyruvate. When we carried out this assay using L-phenylalanine as the amine donor, we noticed that it seems, as seen in Figure 4, as though AATA was successfully able to undergo the enzymatic cycle and complete the transfer of the amine group. The evidence that points to this is seen by the noticeable shift in absorbance peak from ~411 nm (black line) when there is no amine donor to 330 nm upon immediate addition of the amine donor (red line), indicating the release of the internal aldimine to form an external aldimine. After around 30 minutes, this peak shifts slightly to the left at around ~314 nm indicating the protonation of the external aldimine to form PMP (blue line). Upon adding phenylpyruvate, we noticed that the peak at 314 nm persists, however, at the same time, there is a reformation of a peak in the ~420 nm range (green line). This is indicative of the reformation of an internal aldimine which is a result of the enzymatic cycle being complete by transfer of an amine group to phenylpyruvate. The persistence of the peak at 314 nm can be attributed to the availability of extra PMP that didn’t transfer an amine group. While these trends indicate that AATA showed enzymatic activity, it should be noted that ideally, the reformation of a peak at 411 nm at the end of the cycle (green line) has a higher absorbance than the peak at ~ 314 nm. The trends that support the enzymatic activity of AATA are based on a qualitative analysis of the absorbance and for more conclusive results, methods such as mass spectrometry should be employed for confirmation.

Enzymatic activity of AATA with pyruvate

As seen in Figure 5, similar to the results with phenylpyruvate, the first three steps of the enzymatic cycle are correctly represented by peaks at 411 nm (black line), 330 nm (red line) and 314 nm (blue line) as indication of amine donor interactions with the transaminase. However, upon addition of the amine acceptor (green line), there is no change in absorbance indicating that there was no transfer of amine group. This could also be attributed to factors such as substrate concentration and assay conditions and therefore confirmatory tests like mass-spectrometry should be conducted before concluding the lack of enzymatic activity.

Enzymatic activity of AATA with 3,5-diiodo-phenylpyruvic acid

Similar to the results with phenylpyruvate, the first three steps of the enzymatic cycle seen in Figure 6 are correctly carried out as represented by peaks at 411 nm (black line), 330 nm (red line) and 314 nm (blue line) as indication of amine donor interactions with the transaminase. In the case of our test acceptor, we again see the reformation of a peak at ~420 nm (green line), similar to phenylpyruvate. This indicates that the amine group was successfully transferred and PLP was internally bound again. However, the red shift of this peak to be further away from the expected peak at ~411 nm could be caused by reasons other than instrumental error. Initial UV-VIS spectroscopy results indicate that AATA has the potential to accept 3,5,-diiodo-phenylpyruvic acid but this should be confirmed with more thorough analysis of the product formed.To make sure that the final product we aimed to synthesize, 3,5-diiodo-L-tyrosine was indeed produced, the assay should be visualized by mass spectrometry.

Summary of UV-VIS Results

Our UV-VIS results using AATA across three amine acceptors show some key inferences.

1. The interaction between AATA and the amine donor, L-phenylalanine, is consistent across all trials. This is seen in Figure 7A by the decrease in the ratio of absorbance between 411 nm and 330 nm upon adding the amine donor. The ratio being above 1 without the donor (black bar) and below 1 (red bar) shows the switch in absorbance with 411 nm being initially high but upon addition of the donor, the absorbance at 330 nm is greater. This is further supported by the decrease in absorbance ratio at 330 nm and 314 nm seen in Figure 7B. While the decrease in the absorbance ratio doesn’t indicate that the absorbance at 330 nm is lesser than 314 nm after waiting 30 minutes (blue bar) to indicate the formation of PMP, this leftward shift in absorbance is more clearly seen in the spectrum results in Figures 4,5 and 6. We suspect this to be due to a red shift in the spectrum after adding any substrate to the reaction mixture. (For instance, all the peaks observed after adding any substrates appear at 12 nm more than the expected values. These observations are consistent with other experiments employing similar methodology.)

2. While there is a shift in absorbance ratios from before and after adding phenylpyruvate and 3,5,-diiodo-phenylpyruvic acid (test acceptor), no change in absorbance is detected after adding pyruvate. To account for the red shift explained above, the absorbances at 326 nm and 423 nm (12 nm more than expected values) were used to analyze the change in absorbance ratio in Figure 7C. From Figure 7C, we can see that while the change doesn’t cause the absorbance at 423 nm to be greater than the absorbance at 326 nm, the decrease in the ratio indicates the formation of a peak/ increase in absorbance at 423 nm upon addition of the amine acceptor (green bar).

A qualitative analysis of the data collected supports the enzymatic activity of AATA (specifically with phenylpyruvate) and demonstrates potential for reaction with our test amine acceptor, 3,5,-diiodo-phenyl pyruvic acid to produce “minithryoxine”. Mass spectrometry would be needed to confirm the preliminary results presented here.