Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA.
J Neural Transm (Vienna). 2011 Jul;118(7):1101-9. doi: 10.1007/s00702-010-0559-4. Epub 2010 Dec 29.
In order to identify the ligands coordinating with copper in lysyl oxidase, the enzyme was expressed in an E. coli expression system and active enzyme obtained after refolding in the presence of Cu(II). The five histidines found in the putative copper-binding region were sequentially mutated to alanines and the enzymatic activities of the resultant mutants were monitored, together with the copper content, the CD and fluorescence spectra, and the redox-cycling activity. The spectroscopic results show that in all cases the protein folded correctly but that the copper-content, enzymatic activity, and redox-cycling ability depended on the mutation. One mutant was fully functional, and two others completely lacked copper, the lysyl tyrosyl quinone (LTQ) cofactor, and activity. A fourth incorporated copper but lacked LTQ and enzymatic activity. The remaining mutant incorporated copper and had redox-cycling activity but no enzymatic activity. The results suggest that three of the five histidines in the putative copper-binding domain, H292, H294, H296, are the copper ligands and essential to the formation of LTQ. A fourth, H289, is not involved in LTQ formation or activity, while a fifth, H303, is suggested to be a general base in the catalytic mechanism.
为了鉴定赖氨酰氧化酶中与铜配位的配体，该酶在大肠杆菌表达系统中表达，并在存在 Cu(II) 的情况下通过重折叠获得活性酶。依次突变假定的铜结合区域中的 5 个组氨酸为丙氨酸，并监测所得突变体的酶活性、铜含量、CD 和荧光光谱以及氧化还原循环活性。光谱结果表明，在所有情况下，蛋白质均正确折叠，但铜含量、酶活性和氧化还原循环能力取决于突变。一种突变体具有完全功能，另外两种则完全缺乏铜、赖氨酰酪氨酸醌（LTQ）辅因子和活性。第四个突变体结合了铜，但缺乏 LTQ 和酶活性。其余的突变体结合了铜，具有氧化还原循环活性但没有酶活性。结果表明，假定的铜结合域中的 5 个组氨酸中的 3 个，即 H292、H294 和 H296，是铜配体，对 LTQ 的形成至关重要。第四个，H289，不参与 LTQ 的形成或活性，而第五个，H303，被认为是催化机制中的一个通用碱。
