Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Biochemistry. 2010 Jan 19;49(2):386-92. doi: 10.1021/bi9017945.
In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine to spermidine and 3-aminopropanal. Mechanistic studies have been conducted with the recombinant human enzyme. The initial velocity pattern in which the ratio between the concentrations of spermine and oxygen is kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with a limiting value (k(3)) of 49 s(-1) and an apparent K(d) value of 48 microM at pH 8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s(-1), comparable to the k(cat) value of 6.6 s(-1). The kinetics of the oxidative half-reaction depend on the aging time after the spermine and enzyme are mixed in a double-mixing experiment. At an aging time of 6 s, the reaction is monophasic with a second-order rate constant of 4.2 mM(-1) s(-1). At an aging time of 0.3 s, the reaction is biphasic with two second-order constants equal to 4.0 and 40 mM(-1) s(-1). Neither is equal to the k(cat)/K(O(2)) value of 13 mM(-1) s(-1). These results establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with oxygen. The k(cat)/K(M) value for spermine exhibits a bell-shaped pH profile, with an average pK(a) value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k(3) shows a pK(a) value of 7.4 for a group that must be unprotonated. The pK(i)-pH profiles for the competitive inhibitors N,N'-dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the inhibitors and the unprotonated form of an amino acid residue with a pK(a) of approximately 7.4 in the active site are preferred for binding.
在哺乳动物细胞中，黄素蛋白亚精胺氧化酶（SMO）催化亚精胺氧化为精脒和 3-氨基丙醛。已经对重组人酶进行了机制研究。在保持亚精胺和氧浓度比恒定的初始速度模式下，建立了乒乓稳态动力学模式。在没有氧的情况下，SMO 被亚精胺还原是两相的。快速相的速率常数随底物浓度而变化，在 pH8.3 时，其极限值（k(3)）为 49 s(-1)，表观 K(d)值为 48 μM。慢步骤的速率常数与亚精胺浓度无关，值为 5.5 s(-1)，与 6.6 s(-1)的 k(cat)值相当。氧化半反应的动力学取决于在双混合实验中混合亚精胺和酶后的老化时间。在老化时间为 6 s 时，反应是单相的，二级反应常数为 4.2 mM(-1) s(-1)。在老化时间为 0.3 s 时，反应是两相的，两个二级常数等于 4.0 和 40 mM(-1) s(-1)。两者都不等于 13 mM(-1) s(-1)的 k(cat)/K(O(2))值。这些结果表明，还原黄素中间产物与氧反应存在不止一条途径。亚精胺的 k(cat)/K(M)值呈现钟形 pH 曲线，平均 pK(a)值为 8.3。该曲线与具有三个带电荷氮的活性亚精胺一致。k(3)的 pH 曲线显示出 pK(a)值为 7.4 的基团必须是非质子化的。竞争性抑制剂 N,N'-二苄基丁烷-1,4-二胺和精脒的 pK(i)-pH 曲线表明，抑制剂的全质子化形式和活性位点中 pK(a)约为 7.4 的氨基酸残基的非质子化形式是首选的结合形式。
