Characterization of pectin methyltransferase from soybean hypocotyls.
Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycine max Merr.). The enzyme was maximally active at pH 6.8 and 35-40 degrees C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 microg x ml(-1), respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [14C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo.
Ishikawa
M
M
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Urawa, Japan.
Kuroyama
H
H
Takeuchi
Y
Y
Tsumuraya
Y
Y
D002473:Cell Wall / Q000737:chemistry; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D002854:Chromatography, Paper; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000201:enzymology*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D010368:Pectins / Q000378:metabolism; D053830:Plant Viral Movement Proteins; D011134:Polysaccharides / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D053830:Plant Viral Movement Proteins; D011134:Polysaccharides; D014764:Viral Proteins; D012436:S-Adenosylmethionine; D010368:Pectins