S. Beeckmans et E. Vandriessche, PIG-HEART FUMARASE CONTAINS 2 DISTINCT SUBSTRATE-BINDING SITES DIFFERING IN AFFINITY, The Journal of biological chemistry, 273(48), 1998, pp. 31661-31669
A eukaryotic fumarase is for the first time unequivocally shown to con
tain two distinct substrate-binding sites. Pig heart fumarase is a tet
rameric enzyme consisting of four identical subunits of 50 kDa each. B
esides the true substrates L-malate and fumarate, the active sites (si
tes A) also bind their analogs D-malate and oxaloacetate, as well as t
he competitive inhibitor glycine, The additional binding sites (sites
B) on the other hand also bind the substrates and their analogs D-mala
te and oxaloacetate, as well as L-aspartate which is not an inhibitor.
Depending on the pH, the affinity of sites B for ligands (K-d being i
n the millimolar range) is 1-2 orders of magnitude lower than the affi
nity of sites A (of which hh is in the micromolar range). However, sat
urating sites B results in an increase in the overall activity of the
enzyme. The benzenetetracarboxyl compound pyromellitic acid displays v
ery special properties. One molecule of this ligand is indeed able to
bind into a site A and a site B at the same time, Four molecules of py
romellitic acid were found to bind per molecule fumarase, and the affi
nity of the enzyme for this ligand is very high (K-d = 0.6 to 2.2 mu M
, depending on the pH). Experiments with this ligand turned out to be
crucial in order to explain the results obtained. An essential tyrosin
e residue is found to be located in site A, whereas an essential methi
onine residue resides in or near site B, Upon limited proteolysis, a p
eptide of about 4 kDa is initially removed, probably at the C-terminal
side; this degradation results in inactivation of the enzyme. Small l
ocal conformational changes in the enzyme are picked up by circular di
chroism measurements in the near-UV region. This spectrum is built up
of two tryptophanyl triplets, the first one of which is modified upon
saturating the active sites (A), and the second one upon saturating th
e low affinity binding sites (B).