Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Biochemistry. 2012 Jun 19;51(24):4888-97. doi: 10.1021/bi300517s. Epub 2012 Jun 5.
The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N(1)-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N(1)-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k(cat)/K(O2) value changes very little upon mutation with N(1)-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k(cat)/K(M)-pH profiles with N(1)-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK(a) values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK(a) as wild-type Fms1, about ∼7.4; this pK(a) is assigned to the substrate N4. The k(cat)/K(O2)-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK(a) of about ∼6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k(cat) value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 Å. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure.
酿酒酵母黄素蛋白氧化酶 Fms1 催化腐胺和 N(1)-乙酰腐胺氧化为亚精胺和 3-氨基丙醛或 N-乙酰-3-氨基丙醛。在 Fms1 的活性中心，His67 与聚胺底物形成氢键。该残基在其他多胺氧化酶中也保守。已对 H67Q、H67N 和 H67A Fms1 的催化特性进行了表征，以评估该残基在催化中的作用。以腐胺和 N(1)-乙酰腐胺作为胺底物时，黄素还原的一级速率常数降低 2-3 个数量级，其中 H67Q 突变的影响最小，H67N 的影响最大。当 N(1)-乙酰腐胺作为胺底物时，突变对 k(cat)/K(O2)值的影响很小，而当使用腐胺时，k(cat)/K(M)-pH 曲线仅降低一个数量级。所有突变体的 N(1)-乙酰腐胺的 k(cat)/K(M)-pH 曲线呈钟形；与野生型酶的曲线相似，表明 His67 不是两个 pK(a)值的原因。所有突变酶黄素还原速率常数的 pH 曲线也显示相同的 pK(a)值，约为 7.4；该 pK(a)值分配给底物 N4。野生型 Fms1 和 H67A 酶的 k(cat)/K(O2)-pH 曲线均显示约为 6.9 的 pK(a)值；这表明 His67 不是这种 pH 行为的原因。对于 H67Q、H67N 和 H67A 酶，当单个残基质子化时，k(cat)值会降低，就像野生型酶一样。已经确定了 H67Q Fms1 的结构，分辨率为 2.4 Å。结构表明，突变破坏了活性中心的氢键网络，表明 His67 对与底物的直接相互作用以及维持整个活性中心结构都很重要。
