Dataset Information

Quaternary Structure of the Tryptophan Synthase ?-Subunit Homolog BX1 from Zea mays.

ABSTRACT: BX1 from Zea mays (zmBX1) is an enzyme of plant secondary metabolism that generates indole for the synthesis of plant defensins. It is a homologue of the tryptophan synthase ?-subunit, TrpA. Whereas TrpA itself is a monomer in solution, zmBX1 is dimeric, confirmed in our work by native MS. Using cross-linking and mutagenesis, we identified the physiological dimerization interface of zmBX1. We found that homodimerization has only minor effects on catalysis and stability. A comparison of the zmBX1-zmBX1 homodimer and zmTrpA-zmTrpB heterodimer interfaces suggest that homodimerization in zmBX1 might, at an early point in evolution, have served as a mechanism to exclude the interaction with the tryptophan synthase ?-subunit (zmTrpB), marking its transition from primary to secondary metabolism.

SUBMITTER: Norris A

PROVIDER: S-EPMC7313238 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from <i>Zea mays</i>.

Norris Andrew A Busch Florian F Schupfner Michael M Sterner Reinhard R Wysocki Vicki H VH

Journal of the American Society for Mass Spectrometry 20200113 2

BX1 from <i>Zea mays</i> (zmBX1) is an enzyme of plant secondary metabolism that generates indole for the synthesis of plant defensins. It is a homologue of the tryptophan synthase α-subunit, TrpA. Whereas TrpA itself is a monomer in solution, zmBX1 is dimeric, confirmed in our work by native MS. Using cross-linking and mutagenesis, we identified the physiological dimerization interface of zmBX1. We found that homodimerization has only minor effects on catalysis and stability. A comparison of th ...[more]

PMID: 31933363

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Project description: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.

Dataset Information

Quaternary Structure of the Tryptophan Synthase ?-Subunit Homolog BX1 from Zea mays.

Publications

Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from <i>Zea mays</i>.

Similar Datasets

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