Williamson P R, Kittler J M, Thanassi J W, Kagan H M
Biochem J. 1986 Apr 15;235(2):597-605. doi: 10.1042/bj2350597.
Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.
以往的研究表明，磷酸吡哆醛（PLP）在赖氨酰氧化酶中起辅助因子的作用，该酶可生成弹性蛋白和胶原蛋白中赖氨酸衍生交联的肽基醛前体。本研究探讨了纯化的牛主动脉赖氨酰氧化酶中羰基部分的性质。尽管通过高效液相色谱法（h.p.l.c.）显示，PLP依赖性酶天冬氨酸转氨酶的相应制剂可产生PLP二硝基苯腙，但从赖氨酰氧化酶中无法分离出该衍生物。在用硼氢化钠（NaBH4）还原酶后对赖氨酰氧化酶进行PLP分析，该过程可将PLP - 蛋白质醛亚胺转化为稳定的5'-磷酸吡哆醛功能，在使用针对该表位的单克隆抗体进行的测试中也呈阴性。苯肼对赖氨酰氧化酶有竞争性抑制作用，在37℃下随着时间的推移抑制作用变得不可逆，一级失活速率常数为0.4 min-1，抑制常数（KI）为1 μM。[14C]苯肼以一种方式共价结合到酶中，该方式可被特异性活性位点抑制剂β-氨基丙腈对酶的预先修饰所阻止，并且与功能性活性位点含量相关。酶结合的苯肼的化学稳定性超过了PLP与蛋白质之间连接预期的稳定性。赖氨酰氧化酶的苯肼衍生物的吸收光谱与PLP的苯腙明显不同。结论是，赖氨酰氧化酶含有一种与PLP不同的羰基辅助因子，并且通过稳定的化学键与酶结合。
