A kinetic and stereochemical investigation of the role of lysine-32 in thephenylpyruvate tautomerase activity catalyzed by macrophage migration inhibitory factor
Wh. Johnson et al., A kinetic and stereochemical investigation of the role of lysine-32 in thephenylpyruvate tautomerase activity catalyzed by macrophage migration inhibitory factor, BIOCHEM, 38(48), 1999, pp. 16024-16033
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.