Schuster B, Rétey J
Lehrstuhl Biochemie im Institut für Organische Chemie, Universität Karlsruhe, Germany.
Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8433-7. doi: 10.1073/pnas.92.18.8433.
Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their Vmax for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the beta-HSi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.
来自欧芹的苯丙氨酸解氨酶（EC 4.3.1.5）在翻译后会发生修饰，其Ser-202脱水形成催化必需的脱氢丙氨酸辅基。通过定点诱变将Ser-202的密码子替换为丙氨酸和苏氨酸的密码子。这些突变体以及重组野生型酶在用硼氢化钠处理后，以L-苯丙氨酸为底物时几乎没有活性，但能催化L-4-硝基苯丙氨酸的脱氨反应，L-4-硝基苯丙氨酸也是野生型酶的底物。尽管这些突变体与L-4-硝基苯丙氨酸的反应速度比野生型酶慢约20倍，但其对L-4-硝基苯丙氨酸的Vmax比对L-苯丙氨酸高两个数量级。与作为不良底物的L-酪氨酸不同，苯丙氨酸解氨酶能以与L-苯丙氨酸相当的速率将DL-3-羟基苯丙氨酸（DL-m-酪氨酸）转化。这些结果提示了一种机制，其中关键步骤是辅基对苯环2位或6位的亲电攻击。在生成的碳正离子中，β-HSi原子的活化方式与在硝基类似物中相似。随后氨的消除，伴随着芳环和辅基的恢复，完成催化循环。
