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Internal dynamics and ionization states of the macrophage migration inhibitory factor: comparison between wild-type
T A Soares1, R D Lins, T P Straatsma
1Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, Zurich, Switzerland. tsoares@igc.phys.chem.ethz.ch
Abstract:
The macrophage migration inhibitory factor (MIF) is a cytokine that shares a common structural architecture and catalytic strategy with three isomerases: 4-oxalocrotonate tautomerase, 5-carboxymethyl-2-hydroxymuconate isomerase, and D-dopachrome tautomerase. A highly conserved N-terminal proline acts as a base-acid during the proton transfer reaction catalyzed by these enzymes. Such unusual catalytic strategy appears to be possible only due to the N-terminal proline pK(a) shifted to 5.0-6.0 units. Mutations of this residue result in a significant decrease of the catalytic activity of MIF. Two hypotheses have been proposed to explain the catalytic inefficiency of MIF: the lower basicity of primary amines with regard to secondary ones and the increased flexibility resulting from the replacement of a proline by residues like glycine. To investigate that, we have performed molecular dynamics simulations of MIF wild-type and its mutant P1G, as well as calculated the protonation properties of several mutant forms. It was found that the N-terminal glycine does not show larger fluctuations compared to proline, but the former residue is more exposed to the solvent throughout the simulations. The apparent pK(a) of these residues displays very little change (as expected from the structural rigidity of MIF) and is not significantly affected by the surrounding ionizable residues. Instead, the hydrophobic character of the active site seems to be the main factor in determining the pKa of the N-terminal residue and the catalytic efficiency of MIF.
Insights
Macrophage migration inhibitory factor (MIF) uses N-terminal proline for catalysis. Simulations show active site hydrophobicity, not flexibility, dictates MIF
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Macrophage migration inhibitory factor (MIF) is a cytokine with isomerase activity.
- MIF shares catalytic strategies with tautomerase enzymes, utilizing an N-terminal proline residue.
- The N-terminal proline's unique pKa (5.0-6.0) is crucial for its catalytic function.
Purpose of the Study:
- Investigate the catalytic inefficiency of MIF mutants.
- Differentiate between two hypotheses for MIF's reduced activity: amine basicity/flexibility vs. active site properties.
- Determine the factors influencing the N-terminal residue's pKa and MIF's catalytic efficiency.
Main Methods:
- Molecular dynamics simulations of wild-type MIF and P1G mutant.
- Calculation of protonation properties for various MIF mutant forms.
- Analysis of residue fluctuations, solvent exposure, and active site characteristics.
Main Results:
- N-terminal glycine in the P1G mutant did not exhibit increased fluctuations compared to proline.
- Glycine was more solvent-exposed than proline in simulations.
- Apparent pKa values showed minimal change and were not significantly affected by surrounding residues.
- Active site hydrophobicity emerged as the primary determinant of N-terminal residue pKa and catalytic efficiency.
Conclusions:
- The catalytic inefficiency of MIF mutants is not primarily due to increased flexibility or altered amine basicity.
- The hydrophobic nature of the MIF active site plays a critical role in modulating the N-terminal residue's pKa.
- Hydrophobicity is the key factor governing the catalytic efficiency of MIF.