Sato F, Tsujita T, Katagiri Y, Yoshida S, Yamada Y
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Eur J Biochem. 1994 Oct 1;225(1):125-31. doi: 10.1111/j.1432-1033.1994.00125.x.
S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (norcoclaurine 6-O-methyltransferase), which catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 6-hydroxyl group of 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl)methyl]-6,7- isoquinolinediol (norcoclaurine), was purified from cultured Coptis japonica cells and its enzymic properties were characterized. Purified norcoclaurine 6-O-methyltransferase had apparent pI 4.7, a native molecular mass of 95 kDa (determined by gel filtration) and subunit molecular mass of 40 kDa (SDS/PAGE). The enzyme did not require a divalent cation for activity, and the addition of Fe2+, Cu2+, Co2+, Zn2+, Mn2+, or Ni2+ at 5 mM severely inhibited enzyme activity. Neither p-chloromercuribenzoate, N-methylmaleimide nor iodoacetamide inhibited enzyme activity at 1 mM. 5,6-Dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]qu inolizinium (berberine, the end-product of the biosynthetic pathway in which norcoclurine 6-O-methyltransferase catalyzes an intermediate step) also inhibited the activity by 50% at 10 mM. Norcoclaurine 6-O-methyltransferase methylated both (S)-norcoclaurine and (R)-norcoclaurine and (R,S)-norlaudanosoline. Further characterization of substrate-saturation kinetics and product inhibition of the purified enzyme indicated that norcoclaurine 6-O-methyltransferase follows a bi-bi ping-pong mechanism with Km values of 2.23 mM and 3.95 mM for (R,S)-norlaudanosoline and S-adenosyl-L-methionine, respectively, while Ki values for S-adenosylhomocysteine versus S-adenosyl-L-methionine and (R,S)-norlaudanosoline were 2.1 mM and 0.18 mM, respectively.
S-腺苷-L-甲硫氨酸：去甲乌药碱6-O-甲基转移酶（去甲乌药碱6-O-甲基转移酶）催化S-腺苷-L-甲硫氨酸的S-甲基转移至1,2,3,4-四氢-1-[(4-羟基苯基)甲基]-6,7-异喹啉二醇（去甲乌药碱）的6-羟基上，该酶从黄连培养细胞中纯化得到，并对其酶学性质进行了表征。纯化的去甲乌药碱6-O-甲基转移酶表观pI为4.7，天然分子量为95 kDa（通过凝胶过滤法测定），亚基分子量为40 kDa（SDS/PAGE）。该酶活性不需要二价阳离子，添加5 mM的Fe2+、Cu2+、Co2+、Zn2+、Mn2+或Ni2+会严重抑制酶活性。1 mM的对氯汞苯甲酸、N-甲基马来酰亚胺或碘乙酰胺均不抑制酶活性。5,6-二氢-9,10-二甲氧基苯并[g]-1,3-苯并二恶唑并[5,6-a]喹嗪鎓（小檗碱，去甲乌药碱6-O-甲基转移酶催化中间步骤的生物合成途径的终产物）在10 mM时也能抑制50%的活性。去甲乌药碱6-O-甲基转移酶可使(S)-去甲乌药碱、(R)-去甲乌药碱和(R,S)-去甲劳丹碱甲基化。对纯化酶的底物饱和动力学和产物抑制的进一步表征表明，去甲乌药碱6-O-甲基转移酶遵循双底物双产物乒乓机制，对(R,S)-去甲劳丹碱和S-腺苷-L-甲硫氨酸的Km值分别为2.23 mM和3.95 mM，而S-腺苷同型半胱氨酸对S-腺苷-L-甲硫氨酸和(R,S)-去甲劳丹碱的Ki值分别为2.1 mM和0.18 mM。
