2tpl

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{{STRUCTURE_2tpl| PDB=2tpl | SCENE= }}
{{STRUCTURE_2tpl| PDB=2tpl | SCENE= }}
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'''TYROSINE PHENOL-LYASE FROM CITROBACTER INTERMEDIUS COMPLEX WITH 3-(4'-HYDROXYPHENYL)PROPIONIC ACID, PYRIDOXAL-5'-PHOSPHATE AND CS+ ION'''
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===TYROSINE PHENOL-LYASE FROM CITROBACTER INTERMEDIUS COMPLEX WITH 3-(4'-HYDROXYPHENYL)PROPIONIC ACID, PYRIDOXAL-5'-PHOSPHATE AND CS+ ION===
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==Overview==
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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., &amp; Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.
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{{ABSTRACT_PUBMED_9174368}}
==About this Structure==
==About this Structure==
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[[Category: Plp-dependent enzyme]]
[[Category: Plp-dependent enzyme]]
[[Category: Pyridoxal phosphate]]
[[Category: Pyridoxal phosphate]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Jul 27 19:28:28 2008''

Revision as of 16:28, 27 July 2008

Image:2tpl.png

Template:STRUCTURE 2tpl

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

Template:ABSTRACT PUBMED 9174368

About this Structure

2TPL is a Single protein structure of sequence from Citrobacter freundii. 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

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