2TPL

TYROSINE PHENOL-LYASE FROM CITROBACTER INTERMEDIUS COMPLEX WITH 3-(4'-HYDROXYPHENYL)PROPIONIC ACID, PYRIDOXAL-5'-PHOSPHATE AND CS+ ION

2TPL の概要

• エントリーDOI: 10.2210/pdb2tpl/pdb
• 分子名称: TYROSINE PHENOL-LYASE, CESIUM ION, HYDROXYPHENYL PROPIONIC ACID, ... (4 entities in total)
• 機能のキーワード: lyase, plp-dependent enzyme, pyridoxal phosphate
• 由来する生物種: Citrobacter freundii
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 103905.73

構造登録者

Antson, A.A.,Demidkina, T.V.,Wilson, K.S. (登録日: 1997-01-23, 公開日: 1997-04-01, 最終更新日: 2024-04-03)

主引用文献

Sundararaju, B.,Antson, A.A.,Phillips, R.S.,Demidkina, T.V.,Barbolina, M.V.,Gollnick, P.,Dodson, G.G.,Wilson, K.S.
The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity.
Biochemistry, 36:6502-6510, 1997

PubMed Abstract: The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

PubMed: 9174368
DOI: 10.1021/bi962917+

実験手法

X-RAY DIFFRACTION (2.5 Å)