Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.1.22 (cystathionine beta-synthase)
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Human cystathionine beta-synthase is one of two key enzymes involved in intracellular metabolism of homocysteine. It catalyzes a beta-replacement reaction in which the thiolate of homocysteine replaces the hydroxyl group of serine to give the product, cystathionine. The enzyme is unusual in its dependence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensation reaction that is catalyzed; however the function of the heme in this protein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Soret peak of ferric cystathionine beta-synthase is at 428 nm and shifts to approximately 395 nm upon addition of the thiol chelator, mercuric chloride. This is indicative of 6-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride treatment of the enzyme results in conversion of the rhombic EPR signal to a g = 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine beta-synthase is 6-coordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consistent with the presence of 5 nitrogens and 1 sulfur ligand. Together, these data support assignment of the axial ligands as cysteinate and imidazole in ferric cystathionine beta-synthase.
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PMID:Characterization of the heme in human cystathionine beta-synthase by X-ray absorption and electron paramagnetic resonance spectroscopies. 1095 45

Cystathionine beta-synthase is a key heme and pyridoxal phosphate-dependent enzyme involved in homocysteine metabolism in humans. The role of the recently discovered heme in this protein remains an important open question. The axial ligands to the heme in both the ferrous and ferric states have been assigned as cysteine and histidine residues, respectively. In this study, we have examined the effect of ligation and spin state changes in the heme on the activity of the enzyme. Treatment of the ferric enzyme with HgCl2 results in the conversion of six-coordinate low-spin heme to five-coordinate high-spin heme and is paralleled by a loss of activity. In contrast, treatment of the ferrous enzyme with HgCl2 results in replacement of the cysteine ligand by an unidentified sixth ligand and retention of the six-coordinate state, and is also accompanied by loss of enzyme activity. Treatment of the five-coordinate HgCl2-treated enzyme with thiols, such as homocysteine, results in reversion to a six-coordinate state. Resonance Raman spectroscopy with 34S-labeled enzyme reveals the return of the endogenous thiol ligand under these conditions and rules out direct coordination by the thiolate of homocysteine to the heme.
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PMID:Mercuric chloride-induced spin or ligation state changes in ferric or ferrous human cystathionine beta-synthase inhibit enzyme activity. 1174 63

The response of E. coli to Hg(2+) exposure was investigated using proteomic and metalloproteomic approaches. E. coli was cultured in the LB medium containing HgCl2 and/or selenomethionine. The growth curve of E. coli was measured to estimate the toxicity of Hg(2+) or selenomethionine. After two-dimensional gel electrophoresis (2-DE), distribution of Hg in 2-DE gel was detected with synchrotron radiation X-ray fluorescence (SRXRF) at 4W1B, Beijing Synchrotron Radiation Facility. The proteins with differential expression and those containing Hg were identified with electrospray ionization tandem mass spectrometry (ESI-MS/MS) and peptide mass fingerprinting analysis. The results showed that Hg(2+) can inhibit the growth of E. coli, while supplement of selenomethionine can shorten the lag period induced by Hg(2+), indicating an antagonistic effect of selenomethionine against Hg(2+) toxicity. Mechanistically, Hg was observed to be able to bind pyruvate kinase, a glycolytic enzyme, and modulate the expression of five other proteins, including down-regulation of outer membrane protein W and up-regulation of transcription termination factor rho, cysteine synthase, transaldolase A and alkyl hydroperoxide reductase subunit C. Therefore, our results indicated that mercury may influence osmosis of plasma membrane, antioxidant defense, and glycometabolism of the microorganism. This study demonstrates the high sensitivity of SRXRF in identifying metal-associated proteins compared to conventional proteomic approaches.
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PMID:Cellular response of E. coli upon Hg2+ exposure--a case study of advanced nuclear analytical approach to metalloproteomics. 2377 Nov 80