title: A kinetic and stereochemical investigation of the role of lysine-32 in the phenylpyruvate tautomerase activity catalyzed by macrophage migration inhibitory factor.
abstract: Macrophage migration inhibitory factor (MIF), an immunoregulatory protein, exhibits a phenylpyruvate tautomerase (PPT) activity. The catalytic mechanism of this activity has recently attracted attention in an effort to determine whether there is a relationship between the PPT activity and the role of MIF in various immune and inflammatory processes. One of the active site residues is lysine-32, which is postulated to play two roles: it assists in substrate binding through an interaction with a carboxylate oxygen at C-1 of phenylpyruvate, and it may be partially responsible for lowering the pK(a) of the catalytic base, Pro-1. The role of Lys-32 has been investigated by changing it to an alanine and an arginine and determining the kinetic parameters, the stereoselectivity, the competitive inhibition, and the pH dependence of the resulting K32A- and K32R-catalyzed reactions. For the K32R mutant, these properties are mostly comparable to those determined for the wild type with two exceptions. There is a modest decrease in the stereoselectivity of the reaction and in the binding affinity of the competitive inhibitor, (E)-2-fluoro-p-hydroxycinnamate. These differences are likely due to the increased steric bulk of arginine. For the K32A mutant, there are 11- and 12-fold decreases in k(cat) and k(cat)/K(m), respectively, using phenylenolpyruvate. Part of the decrease in activity can be attributed to the observed increase of 1. 3 units in the pK(a) of Pro-1. It was also found that the loss of the electrostatic interaction did not significantly affect the stereoselectivity of the K32A-catalyzed reaction, although it did result in a decrease in the binding affinity of the competitive inhibitor. The combination of these results indicates that the primary function of Lys-32 in the PPT activity of MIF is to lower the pK(a) of Pro-1. The interactions responsible for the stereoselectivity of the PPT activity were further delineated by examining the wild type- and K32A-catalyzed reactions with an alternate substrate, 2-hydroxy-2,4-pentadienoate, in which the phenyl group of phenylenolpyruvate is replaced with a double bond. The effect of this substitution is moderate as evidenced by the observation that the ketonization of 2-hydroxy-2,4-pentadienoate by the wild type protein is more stereoselective than the K32R-catalyzed ketonization of phenylenolpyruvate but not as stereoselective as the K32A-catalyzed ketonization of phenylenolpyruvate. However, the low degree of stereoselectivity observed for the K32A-catalyzed reaction indicates that an electrostatic interaction between the protein and 2-hydroxy-2, 4-pentadienoate is now crucial.
authors.affiliation: Medicinal Chemistry Division, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
authors.forename: W H
authors.initials: WH
authors.lastname: Johnson
authors.forename: R M
authors.initials: RM
authors.lastname: Czerwinski
authors.forename: S L
authors.initials: SL
authors.lastname: Stamps
authors.forename: C P
authors.initials: CP
authors.lastname: Whitman
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008263:Macrophage Migration-Inhibitory Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008968:Molecular Conformation; D009154:Mutation; D013237:Stereoisomerism
chemical_list: D008263:Macrophage Migration-Inhibitory Factors; D019746:Intramolecular Oxidoreductases; C022346:phenylpyruvate tautomerase; D008239:Lysine