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Planta. 1992 May;187(2):236-41. doi: 10.1007/BF00201945.

Purification and characterization of sinapoylglucose:malate sinapoyltransferase from Raphanus sativus L.

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1
Institut für Pharmazeutische Biologie der Technischen Universität Braunschweig, Mendelssohnstrasse 1, W-3300, Braunschweig, Federal Republic of Germany.

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.

PMID:
24178050
DOI:
10.1007/BF00201945

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