Article Review: Studying the Mechanisms of Phenylpyruvate Tautomerase Through Analyzing the Properties of the Mutants of Asn-97, Pro-1, and Tyr-95

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In the previous four decades, the enzymatic activity of phenylpyruvate tautomerase (PPT) which involves catalyzing the interconversion of the pyruvate’s and phenylpyruvate’s keto and enol isomers. Although it is known that the enzymatic activity of PPT is part of the tyroxine biosynthetic pathway, it is not clear how the activity applies to other tissues of the body. However, the discovery that PPT and the macrophage migration inhibitory factor (MIF) were similar proteins raised the interest on other biological involvement of PPT in the body (PPT activity of MIF). For instance, MIF is involved in glomerulonephritis, rheumatoid arthritis, respiratory distress syndrome in adults, and in the development of sepsis. PPT has also been found to have the same structural homology with 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI) and 4-oxalocrotonate tautomerase (4-OT) which bacterial isomerases proteins. The general base catalyst for the PPT activity of MIF is Pro-1. Further, the general acid catalyst for the activity was Tyr-95 based on its position in the MIF structure. There is no other functional group apart from Tyr-95 located in the area of the MIF structure that can act as a general acid. An active site residue, Asn-97, is involved in the binding of the related hydroxyl and phenolic hydroxyl groups. Succeeding studies have revealed that Pro-1 might act as both general base and general acid in the PPT activity of MIF.

Materials and Methods
Apart from (E)-2-Fluoro-p-hydroxycinnamic acid, all the other materials were purchased. The general method involved performing the high pressure liquid chromatography (HPLC) on an anion-exchange column. The concentrations of the protein was through two methods: Waddell’s method and the bicinchoninic acid assay. Further analysis of the protein was conducted through SDS-PAGE method. The most significant method involved the monitoring or the PPT activity of MIF which was conducted through the ketonization process using the sodium phosphate buffer. Competitive inhibition process was then carried to allow the kinetic data collection. The data collection was conducted using a spectrophotometer. Other activities involved DNA sequencing, fitting kinetic data through non-linear regression analysis, and acquiring the electrospray ionization mass. The preparation of the mutants involved the use of gene from a cloned mouse’s MIF. The Y95F and N97A mutants were prepared through an overlap extension polymerase chain reaction. The mutants then required to be purified and overexpressed using a given published procedure. The mass spectrometry process was used to determine the monomeric masses of the mutant’s proteins after the purification process. This was done using the electrospray ionization spectrometry (ESI-MS). A circular dichroism spectroscopy is also carried out on the four mutants and the wild-type protein. The P1A and Y95F MIF mutants were further analyzed for pH rate profiling. The Y95F mutant went a further step where it was crystallized through hanging drop vapor diffusion approach. Finally, the diffraction data was obtained from the spectroscopy methods and used for crystal determination. Computer based minimization processes were further used to refine the structure of Y95F MIF mutant.

Results and Discussion
After the construction, expression, and purification of the four MIF mutants, it was found that the mutation of Pro-1 to alanine or glycine had little impact on Km. However, its impact on kcal was significantly high. On the other hand, the mutation of Tyr-95 and Asn-97 showed insignificant impact on either km or kcal. The mass spectral analysis presented a peak at 114-amino acid molecular mass as expected. This showed that all the proteins had went through posttranslational processing removing the initiating N-formylmethionine. A comparison between the circular dichroism of the mutants and that of the wild type indicated that there were no significant conformational changes after the mutations. The Y95F mutant site structures were compared with MIF which has been complexed. The findings did not show any significant difference in the active site locations. However, two water molecules were observed in the Y95F’s active site. The Y95F mutant also failed to crystallize in the presence of a competitive inhibitor. Plots of log(kcat/km) against pH for the P1A and Y95F MIF mutants had a single ascending limb which levels off.

The experiment indicated that Tyr-95 was not a significant residue of the tautomerization process. This was due to the lack of a considerable difference between the wild type kinetic parameters and those of the Y95F mutant. The finding created doubts over the Tyr-95 mutants role as the general acid catalyst. This shows that there might be an undiscovered physiological reaction in the PPT activity of MIF where Tyr-95 acts as a general acid catalyst. This further indicates that the function of the keto-enol tautomerization is not yet known. Also, the fact that the reaction involved in the process does not require a general acid catalyst might explain its absence thereof. Normally, when the pka of the abstracted proton is within two or three units of pka of the base catalyst, the general acid catalyst is not essential. There could also be a possibility where the Pro-1 functioned as both the general acid and general base catalyst in the PPT activity of MIF. Also, while Lys-32, Pro-1, Asn-97, and Tyr-95 were found in the assigned region, only the mutants in Pro-1 affected the catalysis process. The plots showed that the catalysis process decreased after the mutation. This could be explained by the slower release of the product and the reduced basicity of the primary amine. Limited mobility of the amino-terminal group and the amino-terminal group being pushed out of position by an extra amino acid may explain the reduced activity of the mutant. Finally, since the PPT activity of MIF mechanism is similar to the 4-OT, the three (MIF, 4-OT, and CHMI) can be said to have an identical ancestral protein root.

  • Stamps, S., Taylor, A., Wang, S., Hackert, M., & Whitman C.P. (2000). Mechanism of the Phenylpyruvate Tautomerase Activity of Macrophage Migration Inhibitory Factor: Properties of the P1G, P1A, Y95F, and N97A Mutants†,‡. Biochemistry, 39(32), 9671-9678. doi: 10.1021/bi000373c