2tpl
From Proteopedia
|
TYROSINE PHENOL-LYASE FROM CITROBACTER INTERMEDIUS COMPLEX WITH 3-(4'-HYDROXYPHENYL)PROPIONIC ACID, PYRIDOXAL-5'-PHOSPHATE AND CS+ ION
Overview
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.
About this Structure
2TPL is a Single protein structure of sequence from Citrobacter freundii with and as ligands. Active as Tyrosine phenol-lyase, with EC number 4.1.99.2 Known structural/functional Sites: and . Full crystallographic information is available from OCA.
Reference
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., Sundararaju B, Antson AA, Phillips RS, Demidkina TV, Barbolina MV, Gollnick P, Dodson GG, Wilson KS, Biochemistry. 1997 May 27;36(21):6502-10. PMID:9174368
Page seeded by OCA on Sun Feb 3 10:46:54 2008
