Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
J Biol Chem. 2011 Aug 12;286(32):28170-80. doi: 10.1074/jbc.M111.241836. Epub 2011 Jun 16.
p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C(2)), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ~30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H(2)O(2) elimination. Studies on pH dependence showed that the rate constant of H(2)O(2) elimination in H396N and H396V increased when pH increased with pK(a) >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK(a) >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H(2)O(2) elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H(2)O(2) elimination in S171A and S171T were ~1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H(2)O(2) elimination increased with pH rise and exhibited an approximate pK(a) of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C(2).
对羟基苯乙酸（HPA）3-羟化酶是一种由两个部分组成的黄素依赖性单加氧酶。根据加氧酶部分（C（2））的晶体结构，His-396 距离黄素 C4a 位置 4.5Å，而 Ser-171 距离黄素 N5 位置 2.9Å。我们研究了这两个残基在 C4a-过氧黄素 FMN 中间物稳定性中的作用。结果表明，与野生型酶相比，H396N 的 C4a-过氧黄素 FMN 形成的速率常数降低了约 30 倍，H396A 降低了 100 倍，H396V 降低了 300 倍。突变对随后的 H2O2 消除步骤的影响较小。对 pH 依赖性的研究表明，在 pH 值升高且 pK(a)分别为>9.6 和>9.7 时，H396N 和 H396V 中的 H2O2 消除速率常数增加，与野生型酶（pK(a)>9.4）相似。这些数据表明 His-396 对于 C4a-过氧黄素 FMN 中间物的形成很重要，但不参与 H2O2 的消除。用氧对 Ser-171 突变体的瞬态动力学研究表明，S171A 和 S171T 的 H2O2 消除速率常数分别比野生型高约 1400 倍和 8 倍。用氧对 S171A 的 pH 依赖性研究表明，H2O2 消除的速率常数随 pH 值的升高而增加，并表现出约为 8.0 的近似 pK(a)。这些结果表明，Ser-171 的羟基侧链与黄素 N5 的相互作用对于 C4a-过氧黄素 FMN 的稳定是必需的。与氧反应的双突变体 S171A/H396V 直接形成氧化黄素，而不稳定 C4a-过氧黄素 FMN 中间物，这证实了基于单突变的发现，即 His-396 对于 C（2）中 C4a-过氧黄素 FMN 中间物的形成很重要，而 Ser-171 对于其稳定很重要。
