Structural Mechanism of Enoyl-CoA Hydratase: Three Atoms from a Single Water Are Added in either an E1cb Stepwise or Concerted Fashion
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- 作者：Brian J. Bahnson ; Vernon E. Anderson ; and Gregory A. Petsko
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：February 26, 2002
- 年：2002
- 卷：41
- 期：8
- 页码：2621 - 2629
- 全文大小：416K
- 年卷期：v.41,no.8(February 26, 2002)
- ISSN：1520-4995
文摘
We have determined the crystal structure of the enzyme enoyl-CoA hydratase (ECH) from ratliver with the bound substrate 4-(N,N-dimethylamino)cinnamoyl-CoA using X-ray diffraction data to aresolution of 2.3 Å. In addition to the thiolester substrate, the catalytic water, which is added in thehydration reaction, has been modeled into well-defined electron density in each of the six active sites ofthe physiological hexamer within the crystallographic asymmetric unit. The catalytic water bridges Glu144and Glu164 of the enzyme and has a lone pair of electrons poised to react with C3 of the enzyme-bound
,
-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent ofthe enolase active site. However, unlike enolase, which has a lysine available to donate a proton, thereare no other sources of protons available from other active site residues in ECH. Furthermore, an analysisof the hydrogen-bonding network of the active site suggests that both Glu144 and Glu164 are ionized andcarry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule,leads to a hypothesis that the three atoms from a single water molecule are added across the double bondto form the hydrated product. The structural results are discussed in connection with details of themechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy ofenzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to theprevious interpretation, the structure of the E-S complex together with previously determined kineticisotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.
,-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent ofthe enolase active site. However, unlike enolase, which has a lysine available to donate a proton, thereare no other sources of protons available from other active site residues in ECH. Furthermore, an analysisof the hydrogen-bonding network of the active site suggests that both Glu144 and Glu164 are ionized andcarry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule,leads to a hypothesis that the three atoms from a single water molecule are added across the double bondto form the hydrated product. The structural results are discussed in connection with details of themechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy ofenzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to theprevious interpretation, the structure of the E-S complex together with previously determined kineticisotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.