Lewandowicz A, Jemielity J, Kańska M, Zoń J, Paneth P
Department of Chemistry, Technical University of Lódź (Lodz), Zeromskiego 116, Lódź, 90-924, Poland.
Arch Biochem Biophys. 1999 Oct 15;370(2):216-21. doi: 10.1006/abbi.1999.1402.
The mechanism by which phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the reversible elimination of ammonia from phenylalanine yielding (E)-cinnamic acid has gained much attention in the recent years. Dehydroalanine is essential for the catalysis. It was assumed that this prostetic group acts as the electrophile, leading to a covalently bonded enzyme-intermediate complex with quarternary nitrogen of phenylalanine. Recently, an alternative mechanism has been suggested in which the enzyme-intermediate complex is formed in a Friedel-Crafts reaction between dehydroalanine and orthocarbon of the aromatic ring. Using semiempirical calculations we have shown that these two alternative mechanisms can be distinguished on the basis of the hydrogen secondary kinetic isotope effect when tritium label is placed in the orthopositions. Our calculations indicated also that the kinetic isotope effect measured using ring-labeled d(5)-phenylalanine could not be used to differentiate these alternative mechanisms. Measured secondary tritium kinetic isotope effect shows strong dependence on the reaction progress, starting at the inverse value of k(H)/k(T) = 0.85 for 5% conversion and reaching the normal value of about 1.15 as the conversion increases to 20%. This dependence has been interpreted in terms of a complex mechanism with initial formation of the Friedel-Crafts type intermediate.
近年来，苯丙氨酸解氨酶（PAL，EC 4.3.1.5）催化从苯丙氨酸可逆消除氨生成（E）-肉桂酸的机制备受关注。脱氢丙氨酸是催化所必需的。据推测，这个辅基充当亲电试剂，导致与苯丙氨酸的季氮形成共价键结合的酶-中间体复合物。最近，有人提出了一种替代机制，即酶-中间体复合物是在脱氢丙氨酸与芳环的原碳之间的傅克反应中形成的。我们通过半经验计算表明，当将氚标记置于邻位时，这两种替代机制可以根据氢二级动力学同位素效应来区分。我们的计算还表明，使用环标记的d(5)-苯丙氨酸测量的动力学同位素效应无法用于区分这些替代机制。测得的二级氚动力学同位素效应显示出对反应进程的强烈依赖性，对于5%的转化率，k(H)/k(T)的倒数为0.85，随着转化率增加到20%，该值达到约1.15的正常值。这种依赖性已根据具有傅克型中间体初始形成的复杂机制进行了解释。
