N-acetyl-D-glucosamine-6-phosphate deacetylase:: Substrate activation via a single divalent metal ion
N-acetyl-D-glucosamine-6-phosphate deacetylase:: Substrate activation via a single divalent metal ion
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DOI：
10.1021/bi700543x
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发表时间：
2007-07-10
期刊：
影响因子：
2.9
通讯作者：
Raushel, Frank M.
中科院分区：
文献类型：
--
作者：
Hall, Richard S.;Xiang, Dao Feng;Raushel, Frank M.
那牙是酰胺水解酶超家族的成员，催化N-乙酰基-（D）-葡糖胺-6-磷酸的脱乙酰化。这种酶的催化机制，解决了金属取代衍生物的催化性能的表征，从大肠杆菌与各种底物类似物的那牙。反应机理是感兴趣的，因为发现来自细菌来源的那牙在活性位点中具有一个或两个二价金属离子。这一观察结果表明，有一个分歧，在进化的那牙，并建议有根本性的差异，在机制的细节为底物活化和水解。那牙从E.大肠杆菌通过去除结合到活性位点的锌而失活，并且脱辅基酶在与1当量的Zn 2+、Cd 2+、Co 2+、Mn 2+、Ni 2+或Fe 2+孵育后重新活化。在所提出的催化机理中，反应通过与二价金属离子和His-143的直接相互作用由底物的羰基的极化引发。在酰胺水解酶超家族的所有成员中β链8末端发现的不变天冬氨酸（Asp-273）从金属结合的水分子（或氢氧化物）中提取质子，以促进对底物羰基的水解攻击。形成四面体中间体，然后在质子通过Asp-273转移到离去基团胺后，随着C-N键的断裂而塌陷。D2 O的溶剂同位素效应的缺乏和没有任何变化的动力学常数与溶剂粘度的增加表明，净产品的形成不限于任何显着程度的质子转移步骤或产品的释放。N-三氟乙酰基-（D）-葡糖胺-6-磷酸被那牙水解的速度比相应的N-乙酰基衍生物快26倍。这一结果是一致的形成或崩溃的四面体中间体的速度限制步骤中的催化机制的那牙。
NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-(D)-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogues. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apoenzyme reactivated upon incubation with 1 equiv of Zn2+, Cd2+, Co2+, Mn2+, Ni2+, or Fe2+. In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate (Asp-273) found at the end of beta-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D2O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton-transfer steps or the release of products. N-Trifluoroacetyl-(D)-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA.