Ligand binding on to maize (Zea mays) malate synthase: a structural study

Abstract

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage 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 cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect 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 presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: Comparison with isoform G and implications for structure‐based drug discovery

“…Prior to the structural studies, the results of small angle X-ray scattering experiments suggested ''opening'' or separation of domains so that the active site could sequester substrates from the solvent (Zipper and Durchschlag 1977). Substrateinduced conformational changes were also consistent with circular dichroism data on MS isolated from maize and yeast (Schmid et al 1974;Beeckmans et al 1994). In contrast, data from subsequent NMR studies of apo ecMSG were inconsistent with domain motions but did suggest disorder in a loop (Thr451-Asp455) that could control access to the active site (Tugarinov et al 2002).…”

“…In particular, oxalate does not stimulate the enolization reaction, giving rise to the idea that oxalate prevents the acetyl group from binding. In another study, it was shown for Zea mays malate synthase (an A isoform) that the affinity for acetyl-CoA is increased almost 50-fold when pyruvate is bound in the active site (Beeckmans et al 1994). These data were interpreted to mean that binding of an appropriate 2-keto acid compound is essential for productive binding of acetyl-CoA.…”

Comparative analysis of malate synthase G from Mycobacterium tuberculosis and E. coli: Role of ionic interaction in modulation of structural and functional properties

“…All malate synthases described till date fall broadly in to two major families, isoforms A and G. The 80 kDa monomeric malate synthase isoform G (MSG) has been found exclusively in bacteria whereas, the oligomeric malate synthase isoform A (MSA, 65 kDa per subunit) occurs in plants and several other organisms including prokaryotes. MSG is a magnesium-dependent enzyme [11]. The members of this family of enzymes are structurally based on TIM barrel fold.…”

Rapid purification of malate synthase from cotyledons of Brassica napus L

“…In this process specialized organelles called glyoxysomes are responsible for both the fl-oxidation of fatty acids and the formation of succinate from the resulting acetyl-CoA via the glyoxylate cycle. Malate synthase, a key enzyme for the glyoxylate cycle, catalyzes the condensation of acetyI-CoA and glyoxylate forming L-malate and CoA [2,3]. The enzyme was purified from several plant species [4][5][6][7][8].…”