PI kinetic and ligand binding study on maize (Zea mays) malate synthas
e is presented. It is concluded from kinetic measurements that the enz
yme proceeds through a ternary-complex mechanism. Michaelis constants
(K-m,K-glyoxylate and K-m,K-acetyl-CoA) were determined to be 104 mu M
and 20 mu M respectively. C.d. measurements in the near u.v.-region i
ndicate that a conformational change is induced in the enzyme by its s
ubstrate, glyoxylate. From these studies we are able to calculate the
affinity for the substrate (K-d,K-glyoxylate) as 100 mu M. A number of
inhibitors apparently trigger the same conformational change in the e
nzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of
inhibitors bearing more bulky groups and/or an extra carboxylic acid a
lso induce a conformational change, which is, however, clearly differe
nt from the former one. Limited proteolysis with trypsin results in cl
eavage of malate synthase into two fragments of respectively 45 and 19
kDa. Even when no more intact malate synthase chains are present, the
final enzymic activity still amounts to 30% of the original activity.
If trypsinolysis is performed in the presence of acetyl-CoA, the clea
vage reaction is appreciably slowed down. The dissociation constant fo
r acetyl-CoA (K-d,K-acetyl-CoA) was calculated to be 14.8 mu M when th
e glyoxylate subsite is fully occupied by pyruvate and 950 mu M (= 50
x K-m) when the second subsite is empty. It is concluded that malate s
ynthase follows a compulsory-order mechanism, glyoxylate being the fir
st-binding substrate. Glyoxylate triggers a conformational change in t
he enzyme and, as a consequence, the correctly shaped binding site for
acetyl-CoA is created. Demetallization of malate synthase has no effe
ct on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate
induces the same spectral change in the absence of Mg2+ as in its pres
ence. Nevertheless, malate synthase shows no activity in the absence o
f the cation. We conclude that Mg2+ is essential for catalysis, rather
than for the structure of the enzyme's catalytic site.