Iqbal Aman, Clifton Ian J, Bagonis Maria, Kershaw Nadia J, Domene Carmen, Claridge Timothy D W, Wharton Christopher W, Schofield Christopher J
Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
J Am Chem Soc. 2009 Jan 21;131(2):749-57. doi: 10.1021/ja807215u.
Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates.
酰基酶复合物是许多采用亲核催化的水解酶及相关酶催化反应中的中间体。然而，大多数已报道的关于酰基酶复合物的结构数据是在非催化条件下获得的。最近的红外分析表明，一些酰基酶复合物可能比大多数晶体学分析所暗示的更加灵活。OAT2是N端亲核体（Ntn）水解酶超家族的成员，在一种涉及酰基酶复合物的机制中，催化鸟氨酸和谷氨酸的α-氨基之间乙酰基的可逆转移。我们对鸟氨酸乙酰转移酶（OAT2）进行了溶液状态和晶体状态的生物物理分析。质谱研究确定Thr-181是OAT2催化过程中被乙酰化的残基；(13)C核磁共振分析表明溶液中存在酰基酶复合物。在N-α-乙酰-L-谷氨酸存在的情况下对OAT2进行结晶，得到了Thr-181被乙酰化 的结构；酰基酶复合物的羰基氧位于一个氧负离子孔中，并定位成与Gly-112的主链酰胺NH和Thr-111的醇形成氢键。虽然晶体学分析只揭示了一种结构，但红外光谱表明存在两种不同的酰基酶复合物结构，其羰基伸缩频率分别为1691和1701 cm(-1)。建模研究暗示了两种可能的酰基酶复合物结构，其中一种与晶体结构中观察到的结构以及1691 cm(-1)的红外吸收相关。第二种酰基酶复合物结构只有一个氧负离子孔氢键，被认为产生了1701 cm(-1)的红外吸收。这两种酰基酶复合物结构可以通过Thr-111侧链醇氢从氧负离子孔移开，与被酰化残基Thr-181的主链羰基形成氢键而相互转化。总体而言，结果表明酰基酶复合物结构可能比以前认为的更具动态性，并支持在研究此类中间体时使用综合的生物物理和建模方法。
