University of California, Santa Cruz, Santa Cruz, CA, 95064, USA.
Department of Chemistry and Biochemistry, California State University, Bakersfield, 9001 Stockdale Hwy., Bakersfield, CA, 93311, USA.
Protein J. 2018 Feb;37(1):47-57. doi: 10.1007/s10930-017-9749-3.
Lysyl oxidase (LOX) is a copper-dependent amine oxidase enzyme that catalyzes the formation of crosslinkages of collagen and elastin in connective tissues by oxidative deamination of lysine. Using site-directed mutagenesis, Histidine 303 has been shown to be a key residue that acts as the necessary catalytic base for this enzyme to function properly. Histidine 303 was mutated to isoleucine to remove catalytic activity and to aspartate and glutamate, respectively, in order to provide alternate residues that could act as a general base that could maintain catalytic activity. Overexpression of the H303I mutant yielded 3.9 mg of enzyme per liter of media, the H303D mutant yielded 3.3 mg of enzyme per liter of media, and the H303E mutant yielded 3.0 mg/L of media. Overexpression of wildtype LOX yielded 4.5 mg/L of media, which is a slight improvement from previous yields. Total copper incorporation for H303I was calculated to be 68% and no copper was detected for the H303D and H303E mutants. As LOX requires the self-processed cofactor lysyl tyrosyl quinone (LTQ) for activity, total LTQ content was obtained by reacting the enzyme with phenylhydrazine and using the previously reported extinction coefficient of 15.4 mM/cm. LTQ content for the wildtype enzyme was determined to be 92%, for H303I the total LTQ content was determined to be 36%, and no LTQ was detected for the H303D and H303E mutants. No catalytic activity was detected for any mutants when compared to the wildtype which has a previously reported activity of 0.11 U/mg. Comparison of excitation-emission matrices (EEM) of each of the mutants as compared to the wildtype indicate that all the mutations cause a change in the internal environment of the enzyme, albeit to varying degrees, as evidenced by the observed shifts.
赖氨酰氧化酶（LOX）是一种铜依赖性胺氧化酶，通过赖氨酸的氧化脱氨作用催化结缔组织中胶原蛋白和弹性蛋白的交联形成。通过定点突变，已证明组氨酸 303 是一种关键残基，作为该酶正常发挥功能的必需催化碱。将组氨酸 303 突变为异亮氨酸以去除催化活性，分别突变为天冬氨酸和谷氨酸，以提供可以作为维持催化活性的通用碱的替代残基。H303I 突变体的过表达产生每升培养基 3.9 毫克的酶，H303D 突变体产生每升培养基 3.3 毫克的酶，H303E 突变体产生每升培养基 3.0 毫克的酶。野生型 LOX 的过表达产生每升培养基 4.5 毫克的酶，这比以前的产量略有提高。H303I 的总铜掺入量计算为 68%，而 H303D 和 H303E 突变体未检测到铜。由于 LOX 活性需要自身加工的辅因子赖氨酰酪氨酸醌（LTQ），通过将酶与苯肼反应并用以前报道的 15.4 mM/cm 的消光系数来获得总 LTQ 含量。野生型酶的 LTQ 含量确定为 92%，H303I 的总 LTQ 含量确定为 36%，而 H303D 和 H303E 突变体未检测到 LTQ。与野生型相比，任何突变体都没有检测到催化活性，而野生型的先前报道的活性为 0.11 U/mg。与野生型相比，每种突变体的激发-发射矩阵（EEM）的比较表明，所有突变都导致酶内部环境发生变化，尽管程度不同，这可以从观察到的位移得到证明。
