Speedie M K, Hornemann U, Floss H G
J Biol Chem. 1975 Oct 10;250(19):7819-25.
Two enzymes, tryptophan transaminase and indolepyruvate C-methyltransferase, which are active in the initial steps of the biosynthetic pathway of the antibiotic indolmycin, have been detected and partially purified from cell-free extracts of Streptomyces griseus. The transaminase has been purified 3-fold by ammonium sulfate fractionation. At this stage of purification, it catalyzes the alpha-ketoglutarate and pyridoxal phosphate-dependent transamination of L-tryptophan, 3-methyltryptophan, L-pphenylalanine, and L-tyrosine. The C-methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine to position 3 of the aliphatic side chain of indolepyruvate. No cofactors are required. The C-methyltransferase has been purified 110-fold by ammonium sulfate fractionation, Sephadex G-150 gel filtration, DEAE-Sephadex column chromotography, and Bio-Gel A-5m gel filtration. The enzyme has a broad pH optimum of 7.5 to 8.5. A molecular weight of 55,000 +/- 5,000 has been determined by Sephadex G-200 gel filtration with reference proteins and a molecular weight of 58,000 +/- 8,000 has been determined by sucrose density gradient centrifugation. The enzyme is relatively stable at temperatures of 0-5 degrees but is destroyed by freezing or by heating. The C-methyltransferase is inhibited strongly by the thiol reagents p-chloromercuribenzoate and N-ethylmaleimide. The Zn2+ and Fe2+ chelators 1,10-phenanthroline and 2,2'-bipyridine also inhibit the enzyme activity but EDTA does not. Michaelis-Menten constants have been determined for the 110-fold purified enzyme as 1.2 X 10(-5) M for S-adenosylmethionine and 4.8 X 10(-6) M for indolepyruvate. The enzyme activity in the crude extract is inhibited competitively by indolmycin (Ki equals 2.3 mM) and L-tryptophan (Ki equals 0.17 mM), but these effects are not observed after the enzyme has been passed through the Sephades G-150 column during purification. The crude extract is capable of methylating phenylpyruvate and p-hydroxyphenylpyruvate but this capability is lost upon purification of the indolepyruvate C-methyltransferase activity. No methylation of L-tryptophan occurs under the conditions used.
在抗生素吲哚霉素生物合成途径的起始步骤中发挥作用的两种酶，色氨酸转氨酶和吲哚丙酮酸C - 甲基转移酶，已从灰色链霉菌的无细胞提取物中被检测到并部分纯化。通过硫酸铵分级分离，转氨酶已被纯化了3倍。在纯化的这个阶段，它催化L - 色氨酸、3 - 甲基色氨酸、L - 苯丙氨酸和L - 酪氨酸的α - 酮戊二酸和磷酸吡哆醛依赖性转氨作用。C - 甲基转移酶催化一个甲基从S - 腺苷甲硫氨酸转移到吲哚丙酮酸脂肪族侧链的3位。不需要辅助因子。通过硫酸铵分级分离、Sephadex G - 150凝胶过滤、DEAE - Sephadex柱色谱和Bio - Gel A - 5m凝胶过滤，C - 甲基转移酶已被纯化了110倍。该酶的最适pH范围很宽，为7.5至8.5。通过使用标准蛋白质的Sephadex G - 200凝胶过滤测定其分子量为55,000±5,000，通过蔗糖密度梯度离心测定其分子量为58,000±8,000。该酶在0 - 5摄氏度相对稳定，但会因冷冻或加热而失活。C - 甲基转移酶受到硫醇试剂对氯汞苯甲酸和N - 乙基马来酰亚胺的强烈抑制。锌离子和亚铁离子螯合剂1,10 - 菲咯啉和2,2'-联吡啶也抑制酶活性，但乙二胺四乙酸（EDTA）不会。对于经过110倍纯化的酶，已测定出其米氏常数：S - 腺苷甲硫氨酸为1.2×10⁻⁵M，吲哚丙酮酸为4.×10⁻⁶M。粗提物中的酶活性受到吲哚霉素（抑制常数Ki等于2.3 mM）和L - 色氨酸（抑制常数Ki等于0.17 mM）的竞争性抑制，但在纯化过程中酶通过Sephades G - 150柱后未观察到这些效应。粗提物能够使苯丙酮酸和对羟基苯丙酮酸甲基化，但在纯化吲哚丙酮酸C - 甲基转移酶活性后这种能力丧失。在所使用的条件下，L - 色氨酸不会发生甲基化。
