Expression of the Arabidopsis sterol methyltransferase2 (SMT2) cDNA in Escherichia coli yields a native protein, when purified to homogeneity, has the predicted molecular mass ca. 40 kDa on SDS-PAGE and recognizes native sterols synthesized by Arabidopsis with a Delta(24(25))-bond (cycloartenol; K(m) 35 microM and k(cat) 0.001s(-1)) and Delta(24(28))-bond (24(28)-methylenelophenol; K(m) 28 microM and k(cat) 0.01 s(-1)). Cycloartenol was converted to a single olefinic product-24(28)-methylenecycloartanol whereas 24(28)-methylenelophenol was converted to a mixture of three stereochemically related products with the Delta(24(28))Z-ethylidene, Delta(24(28))E-ethylidene, and Delta(25(27))-24 beta-ethyl side chains. Structural determinants essential to activity were the nucleophilic features at C-3 and C-24. The double bond position in the sterol substrate influenced catalytic efficiency according to the order: side chain, Delta(24(24))<Delta(24(28)) and nucleus, Delta(7)<Delta(8)<Delta(5)=9,19-cyclopropane. The 14 alpha-methyl group was harmful to catalysis, reducing the suitability of cycloartenol as a substrate. On the basis of substrate activity and product distribution, SMT action was probed further using substrate (26,27-dehydrozymosterol: 26,27-DHZ) and intermediate (25-azacycloartenol: 25-AC) analogs of the SMT-catalyzed reactions. 26,27-DHZ was C-methylated to 26-homocholesta-8(9), 23(24)E, 26(26('))-trienol as well as 26-homocholesta-8(9),26(26')-3 beta,24 beta-dienol by SMT2, K(m) of 15 microM, k(cat) of 0.001 s(-1). In addition, 26,27-DHZ acted as a mechanism-based irreversible inhibitor that results in the specific covalent modification of SMT2, exhibiting K(i) of 49 microM, k(inact) of 0.009 s(-1) and partition ratio of 0.11. Substrate protection with zymosterol, 24(28)-methylenelophenol against 26,27-DHZ and similar inhibition of the first and second C(1)-transfer activities by the reversible inhibitor 25-AC of K(i) 20 nM suggested the analogs interacted at the same active site. [28E-2H]- and [28Z-2H]24(28)-methylenelanosterols were paired with AdoMet and differences of 2H-incorporation in the enzyme-generated 24-ethyl olefins supported an antimechanism. The results suggest plant SMT2 has a position-specific substrate specificity for Delta(24(25))-sterols and contains a single active center to catalyze the consecutive C(1)-transfer activities by substrate reaction channels similar to the fungal SMT1.