LIGAND-BINDING ON TO MAIZE (ZEA-MAYS) MALATE SYNTHASE - A STRUCTURAL STUDY

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.