A kinetic and stereochemical investigation of the role of lysine-32 in thephenylpyruvate tautomerase activity catalyzed by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF), an immunoregulatory protein, exhibits a phenylpyruvate tautomerase (PPT) activity. The catalytic mechani sm of this activity has recently attracted attention in an effort to determ ine whether there is a relationship between the PPT activity and the role o f 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( n) of the catalytic base, Pro-1. The role of Lys-32 has been investigated b y changing it to an alanine and an arginine and determining the kinetic par ameters, the stereoselectivity, the competitive inhibition, and the pH depe ndence of the resulting K32A- and K32R-catalyzed reactions. For the K32R mu tant, these properties are mostly comparable to those determined for the wi ld type with two exceptions. There is a modest decrease in the stereoselect ivity of the reaction and in the binding affinity of the competitive inhibi tor, (E)-2-fluoro-p-hydroxycinnamate. These differences are likely due to t he increased steric bulk of arginine. For the K32A mutant, there are 11- an d 1Zfold decreases in k(cat) and k(cat)/k(m), respectively, using phenyleno lpyruvate. Part of the decrease in activity can be attributed to the observ ed 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 ster eoselectivity of the K32A-catalyzed reaction, although it did result in a d ecrease in the binding affinity of the competitive inhibitor. The combinati on of these results indicates that the primary function of Lys-32 in the PP T activity of MIF is to lower the pK(a) of Pro-1. The interactions responsi ble for the stereoselectivity of the PPT activity were further delineated b y examining the wild type- and K32A-catalyzed reactions with an alternate s ubstrate, 2-hydroxy-2,4-pentadienoate, in which the phenyl group of phenyle nolpyruvate is replaced with a double bond. The effect of this substitution is moderate as evidenced by the observation that the ketonization of 2-hyd roxy-2,4-pentadienoate by the wild type protein is more stereoselective tha n the K32R-catalyzed ketonization of phenylenolpyruvate but not as stereose lective as the K32A-catalyzed ketonization of phenylenolpyruvate, However, the low degree of stereoselectivity observed for the K32A-catalyzed reactio n indicates that an electrostatic interaction between the protein and 2-hyd roxy-2,4-pentadienoate is now crucial.