CHARACTERIZATION OF THE ROLE OF THE AMINO-TERMINAL PROLINE IN THE ENZYMATIC-ACTIVITY CATALYZED BY MACROPHAGE-MIGRATION INHIBITORY FACTOR

The cytokine macrophage migration inhibitory factor (MIF) mediates sev eral immune and inflammatory processes through unknown or poorly under stood mechanisms. The protein shares structural homology with two bact erial isomerases, 4-oxalocrotonate tautomerase (4-OT) and 5-(carboxyme thyl)-2-hydroxymuconate isomerase (CHMI), and catalyzes the enolizatio n of phenylpyruvate and the ketonization of (p-hydroxyphenyl)pyruvate. The amino-terminal proline has been identified as the catalytic base in both the 4-OT- and CHMI-catalyzed reactions. MIF also has an amino- terminal proline that has been implicated as a catalytic group in the MIF-catalyzed reaction. To delineate further the role of Pro-1 in the MIF-catalyzed reaction, affinity labeling studies were performed with 3-bromopyruvate (3-BP). The results of this study show that 3-BP acts as an active-site-directed irreversible inhibitor of the enzymatic act ivity and modifies one site per monomeric subunit, The inhibitor, as i ts lactyl derivative, is covalently attached to an 11 residue amino-te rminal fragment, Pro-1 to Arg-ll. The only reasonable site for alkylat ion within this peptide fragment is the amino-terminal proline. Becaus e the pK(a) measured for the pH dependence of k(inact)/K-I (5.7 +/- 0. 2) and that measured for the pH dependence of the k(cat)/K-m for the e nolization of phenylpyruvate (6.0 +/- 0.1) are comparable and in reaso nable agreement with the previously measured pK(a) of Pro-1 (5.6 +/- 0 .1) obtained by its direct titration [Swope, M., Sun H.-W., Blake, P., and Lolls, E. (1998) EMBO J. (in press)], it is concluded that Pro-1 acts as the general base catalyst in the MIF-catalyzed reaction. The s tructural and mechanistic parallels place 4-OT, CHMI, and MIF in a sup erfamily of enzymes related by their ability to catalyze the keto-enol tautomerization of a pyruvyl moiety.