Abstract
1-O-Sinapoyl-β-glucose:l-malate O-sinapoyltransferase (SMT; EC 2.3.1.) from cotyledons of red radish (Raphanus sativus L. var. sativus) was purified to apparent homogeneity with a 2100-fold enrichment and a 4% recovery. Apparent Mrs of 52 and 51, respectively, were determined by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On isoelectric focusing, the SMT resolved into two isoforms which, on SDS-PAGE, showed, slightly different Mrs (SMT I: Mr/isoelectric point = 51/5.75; SMT II: Mr/isoelectric point = 51.5/5.9). The highest activity of SMT was found at pH 6.0 (50% at pH 5.5 and pH 6.5). The temperature maxima in the presence of 10, 50, 100 and 250 mM malate were 22, 30, 35 and 37° C, respectively, with energies of activation of 55, 81, 96 and 121 kJ · mol-1. The enzyme accepted all the hydroxycinnamic acid-glucose esters tested with relative ratios of initial velocity values of 100∶85∶45∶26∶2.6 of 1-O-sinapoyl-, 1-O-feruloyl-, 1-O-caffeoyl-, 1,2-di-O-sinapoyl-, and 1-O-(4-coumaroyl)-β-glucose. It showed an absolute acceptor specificity for l-malate. d-Malate as second acceptor molecule in standard assays with l-malate inhibited the reaction velocity noncompetitively (K i = 215 mM). The substrate saturation curves were not hyperbolic. The data for sinapoylglucose indicated substrate activation; those for l-malate, substrate inhibition. Kinetic analysis suggests a random bi bi mechanism within two ranges of substrate concentrations, with a kinetically preferred pathway via the enzyme-sinapoylglucose complex indicating a slow-transition mechanism. This may be interpreted as hysteretic cooperativity with sinapoylglucose.
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Abbreviations
- IEF:
-
isoelectric focusing
- Mal:
-
l-malate
- pI:
-
isoelectric point
- SinGlc:
-
1-O-sinapoyl-β-glucose
- SinMal:
-
O-sinapoyl-l-malate
- SMT:
-
1-O-sinapoyl-β-glucose: l-malate sinapoyltransferase
- SMT I and SMT II:
-
SMT isoforms isolated after isoelectric focusing
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We thank H. Bisswanger (Physiologisch-chemisches Institut, Universität (Tübingen, FRG) for help on the interpretation of substrate kinetic data and B.E. Ellis (Department of Plant Science, The University of British Columbia, Vancouver, B.C., Canada) for linguistic advice. Support by the Deutsche Forschungsgemeinschaft (Bonn, FRG) and the Fonds der Chemischen Industrie (Frankfurt, FRG) is gratefully acknowledged.
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Gräwe, W., Bachhuber, P., Mock, HP. et al. Purification and characterization of sinapoylglucose:malate sinapoyltransferase from Raphanus sativus L.. Planta 187, 236–241 (1992). https://doi.org/10.1007/BF00201945
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DOI: https://doi.org/10.1007/BF00201945