IDENTIFICATION OF METAL LIGANDS IN CU(II)-INHIBITED CHROMOBACTERIUM-VIOLACEUM PHENYLALANINE-HYDROXYLASE BY ELECTRON-SPIN ECHO ENVELOPE MODULATION ANALYSIS OF HISTIDINE TO SERINE MUTATIONS

被引:18
作者
BALASUBRAMANIAN, S
CARR, RT
BENDER, CJ
PEISACH, J
BENKOVIC, SJ
机构
[1] PENN STATE UNIV, DEPT CHEM, UNIV PK, PA 16802 USA
[2] YESHIVA UNIV ALBERT EINSTEIN COLL MED, DEPT MOLEC PHARMACOL, BRONX, NY 10461 USA
关键词
D O I
10.1021/bi00194a019
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) is known to bind an equivalent of divalent copper. The ''metal-free'' form of the protein is fully active, and Cu(II) is now shown to be an inhibitor of CVPAH rather than an activator of the enzyme [Carr, R. T., and Benkovic, S. J. (1994) Biochemistry 32, 14132-14138]. On the basis of amino acid sequence homology, the metal binding site may be related to those of rat liver PAH and other eukaryotic pterin-dependent hydroxylases, which require Fe(II) for activity. The conserved histidines at that site in CVPAH, histidines 138 and 143, were each mutated to serines. The mutant enzymes H138S and H143S were both catalytically inactive, but still able to bind Cu(II). Binding studies further demonstrated that both mutant enzymes still bind L-phenylalanine. Electron spin echo envelope modulation (ESEEM) studies on each of the mutants showed the presence of only a single copper-coordinating histidine, rather than the two histidine ligands suggested for the wild-type protein. This result supports a model in which Cu(II) is equatorially ligated to only two histidines in the Cu(II)-inhibited protein and allows us to unambiguously assign histidines 138 and 143 as these ligands. That the enzyme is inactive when these histidines are either bound with copper or when replaced with serines suggests that these histidines perform a catalytic function. Possible catalytic roles for these histidines in the hydroxylation mechanism of pterin-dependent monooxygenases are discussed along with potential future applications of the combination of ESEEM with site-directed mutagenesis.
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页码:8532 / 8537
页数:6
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