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

The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a su bstrate analog, 3-(4'hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the sidechain NH1 4.1 A ngstrom from the 4'-OH of the analog. The structure has been deduced a t 2.5 Angstrom resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 Angstrom, b = 143.91 Angstrom, and c = 59 .80 Angstrom. To evaluate the role of Arg 381 in TPL catalysis, we pre pared mutant proteins replacing arginine with alanine (R381A), with is oleucine (R381I), and with valine (R381V). The beta-elimination activi ty of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination ac tivity 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 )/K-m values comparable to those of wild-type TPL. Furthermore, the K- i values for competitive inhibition by L-tryptophan and L-phenylalanin e are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stop ped flow spectroscopic analyses also show that wild-type and mutant pr oteins can bind L-tyrosine and form quinonoid complexes with similar r ate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wi ld-type TPL decreases at high pH values with a pK(a) of 8.4 and is thu s dependent on an acidic group, possibly Arg404, which forms an ion pa ir with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff b ase. R381A TPL shows only a small decrease in k(cat)/K-m for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pK(a) s with an average value of about 7.8. Thus, it is possible that Arg 38 1 is one of the catalytic bases previously observed in the pH dependen ce of k(cat)/K-m of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least p artially responsible for the substrate specificity of TPL.