Osterman A L, Brooks H B, Rizo J, Phillips M A
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Biochemistry. 1997 Apr 15;36(15):4558-67. doi: 10.1021/bi962916h.
The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy. The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A., & Goldsmith, E. J. (1995) Protein Sci. 4, 1291-1304]. Mutation of Arg-277 to Ala increases the K(m) for PLP by 270-fold compared to that of wild-type ODC while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity was measured directly by ultrafiltration; the K(d) for PLP is at least 20-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC also has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necessary for high-affinity PLP binding by ODC. The 31P NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a dianion to both wild-type and R277A ODC. However, the 31P chemical shift for R277A ODC (6.7 ppm) is 0.5 ppm downfield from that observed for the wild-type enzyme, indicating that the environment of the enzyme-bound phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 8.5. Concomitant with these changes are a decreased k(cat) and an altered absorption spectra which arises from bound PLP. PLP bound to wild-type ODC has a 31P chemical shift and a CD signal observable over the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the 31P chemical shift becomes solution-like and the CD signal is abolished. The data suggest that for R277A ODC the rigid PLP binding mode which characterizes the wild-type enzyme is lost at high pH. Thus, multiple interactions between the wild-type active site and PLP maintain the cofactor in a constrained conformation that is essential for efficient catalysis, tempering the consequence of the removal of any single interaction.
通过定点诱变和光谱学方法，对布氏锥虫鸟氨酸脱羧酶（ODC）中的磷酸吡哆醛（PLP）结合位点进行了研究。针对真核ODC结构提出的β/α桶状模型预测，PLP的磷酸基团通过与富含甘氨酸的环（残基235 - 237）相互作用以及与Arg - 277形成盐桥而得以稳定[格里申，N. V.，菲利普斯，M. A.，& 戈德史密斯，E. J.（1995年）《蛋白质科学》4，1291 - 1304]。与野生型ODC相比，将Arg - 277突变为Ala会使PLP的K(m)增加270倍，而在pH 8时k(cat)仅降低2倍。通过超滤直接测量PLP的结合亲和力；在pH 8时，突变酶中PLP的K(d)至少高20倍。此外，R277A ODC对一系列辅因子类似物的结合亲和力也比野生型酶弱。这些结果表明，Arg - 277是ODC高亲和力结合PLP所必需的。ODC的31P NMR光谱表明，磷酸基团以二价阴离子形式以应变构象与野生型和R277A ODC结合。然而，R277A ODC的31P化学位移（6.7 ppm）比野生型酶的化学位移向低场偏移0.5 ppm，这表明突变酶中与酶结合的磷酸基团的环境发生了改变。在高pH下，PLP与野生型和R277A ODC的结合亲和力均较弱，这与一个pK(a)约为8.5的质子化物种的滴定相对应。伴随着这些变化的是k(cat)降低以及由结合的PLP产生的吸收光谱改变。与野生型ODC结合的PLP在整个测试的pH范围（7 - 9）内都具有可观测的31P化学位移和CD信号。相比之下，对于pH 8至9之间的R277A ODC，31P化学位移变得类似于溶液状态，CD信号消失。数据表明，对于R277A ODC，在高pH下失去了表征野生型酶的刚性PLP结合模式。因此，野生型活性位点与PLP之间的多种相互作用将辅因子维持在一种受限构象中，这对于高效催化至关重要，缓和了去除任何单一相互作用的影响。
