Martínez-Rodríguez Sergio, Andújar-Sánchez Montserrat, Clemente Jiménez Josefa María, Jara-Pérez Vicente, Rodríguez-Vico Felipe, Las Heras-Vázquez Francisco Javier
Departamento de Química-Física, Bioquímica y Química Inorgánica, Edificio C.I.T.E. I, Universidad de Almería, La Cañada de San Urbano, Almería 04120, Spain.
Biochimie. 2006 Jul;88(7):837-47. doi: 10.1016/j.biochi.2006.01.012. Epub 2006 Feb 23.
Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.
通过动力学方法和等温滴定量热法（ITC）研究了苜蓿中华根瘤菌CECT 4114 l-N-氨甲酰酶（SmLcar）的纯化定点突变体，其中Glu132、His230、Asn279和Arg292被替换。已证明His230、Asn279和Arg292残基在识别N-氨甲酰-l-α-氨基酸中的重要性。Glu132在底物水解中的作用已得到证实。ITC已证实野生型酶每个单体有两个镍原子，以及两个相等且独立的底物结合位点（每个单体一个）。SmLcar的同源建模支持His87、His194、His386、Glu133和Asp98在金属结合中的重要性。基于ITC测量的结合实验、动力学测定以及活性中心与克鲁维酵母β-丙氨酸合酶和其他酶的同源性，提出了一个全面的反应机制。
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