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 galactosylu ronic acid residues in pectin was found in a membrane preparation of etiola ted hypocotyls from 6-d-old soybean (Glycine max Merr.). The enzyme was max imally 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 mu g . ml(-1), respectively. Atta chment of the methyl group to the carboxyl group of the pectin via ester li nkage was confirmed by analyzing radiolabeled product from incubation of th e enzyme with [C-14]methyl SAM and the acceptor pectin. Size-exclusion chro matography showed that both enzymatic hydrolysis with a pectin methylestera se and a mild alkali treatment (saponification) led to the release of radio active methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low r elative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated i nto their cell wall components by successive extraction with water, EDTA, a nd alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a df-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative ac ceptor substrate, indicating that the enzyme may be responsible for produci ng methyl-esterified pectin in vivo.