Andújar-Sánchez Montserrat, Martínez-Rodríguez Sergio, Heras-Vázquez Francisco Javier Las, Clemente-Jiménez Josefa María, Rodríguez-Vico Felipe, Jara-Pérez Vicente
Dpto. Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, Carretera Sacramento s/n Almería, 04120, España.
Biochim Biophys Acta. 2006 Feb;1764(2):292-8. doi: 10.1016/j.bbapap.2005.11.017. Epub 2005 Dec 21.
Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pure d-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, deltaG, deltaH and DeltaS for the inhibitors L- and D-5-methylthioethyl-hydantoin. The number of active sites is four per enzyme molecule (one per monomer), and the binding of the inhibitor is entropically and enthalpically favoured under the experimental conditions studied. In order to obtain information about amino acids involved in the active site, four different mutants were developed in which cysteines 76 and 181 were mutated to Alanine and Serine. Their behaviour shows that these cysteines are essential for enzyme activity, but only cysteine 76 affects the binding to these inhibitors.
乙内酰脲消旋酶与立体选择性乙内酰脲酶和立体特异性D-氨甲酰酶共同作用，确保了从外消旋化速度较慢的D,L-5-单取代乙内酰脲到光学纯D-氨基酸的完全转化。来自苜蓿中华根瘤菌的乙内酰脲消旋酶在大肠杆菌中表达。然后进行了量热和荧光实验，以获得抑制剂L-和D-5-甲硫基乙基乙内酰脲的热力学结合参数ΔG、ΔH和ΔS。每个酶分子的活性位点数量为四个（每个单体一个），在所研究的实验条件下，抑制剂的结合在熵和焓方面均受到青睐。为了获得有关活性位点中氨基酸的信息，构建了四个不同的突变体，其中半胱氨酸76和181分别突变为丙氨酸和丝氨酸。它们的行为表明，这些半胱氨酸对酶活性至关重要，但只有半胱氨酸76会影响与这些抑制剂的结合。
