Purification and properties of S-adenosyl-L-methionine:L-methionine S-methyltransferase from Wollastonia biflora leaves.
The plant enzyme S-adenosylmethionine:methionine S-methyltransferase (EC 2.1.1.12, MMT) catalyzes the synthesis of S-methylmethionine. MMT was purified 620-fold to apparent homogeneity from leaves of Wollastonia biflora. The four-step purification included fractionation with polyethylene glycol, affinity chromatography on adenosine-agarose, anion exchange chromatography, and gel filtration. Protein yield was about 180 micrograms/kg of leaves. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native gel filtration chromatography were, respectively, 115 and 450 kDa, suggesting a tetramer of 115-kDa subunits. The 115-kDa subunit was photoaffinity labeled by S-adenosyl[3H]methionine. Antibodies raised against W. biflora MMT recognized a 115-kDa polypeptide in partially purified MMT preparations from leaves of lettuce, cabbage, clover, and maize. The pH optimum of W. biflora MMT was 7.2. Kinetic analysis of substrate interaction and product inhibition patterns indicated an Ordered Bi Bi mechanism, with S-adenosylmethionine the first reactant to bind and S-adenosylhomocysteine the last product to be released. The enzyme catalyzed methylation of selenomethionine and ethionine, but not of S-methylcysteine, homocysteine, cysteine, or peptidylmethionine. Tests with other substrate analogs indicated that a free carboxyl group was required for enzyme activity, and that a free amino group was not.
James
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F
Plant Molecular and Cellular Biology Program, University of Florida, Gainesville 32611, USA.
Nolte
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KD
Hanson
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AD
D000345:Affinity Labels; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D011489:Protein Denaturation; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D013379:Substrate Specificity; D014814:Vitamin U / Q000096:biosynthesis* / Q000494:pharmacology
D000345:Affinity Labels; D014814:Vitamin U; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C093871:methionine S-methyltransferase