Moran G R, Entsch B, Palfey B A, Ballou D P
Molecular and Cellular Biology, School of Biological Sciences, University of New England, Armidale, New South Wales 2351, Australia.
Biochemistry. 1999 May 11;38(19):6292-9. doi: 10.1021/bi990021+.
In the crystal structure of native p-hydroxybenzoate hydroxylase, Ser212 is within hydrogen bonding distance (2.7 A) of one of the carboxylic oxygens of p-hydroxybenzoate. In this study, we have mutated residue 212 to alanine to study the importance of the serine hydrogen bond to enzyme function. Comparisons between mutant and wild type (WT) enzymes with the natural substrate p-hydroxybenzoate showed that this residue contributes to substrate binding. The dissociation constant for this substrate is 1 order of magnitude higher than that of WT, but the catalytic process is otherwise unchanged. When the alternate substrate, 2,4-dihydroxybenzoate, is used, two products are formed (2,3,4-trihydroxybenzoate and 2,4, 5-trihydroxybenzoate), which demonstrates that this substrate can be bound in two orientations. Kinetic studies provide evidence that the intermediate with a high extinction coefficient previously observed in the oxidative half-reaction of the WT enzyme with this substrate is composed of contributions from both the dienone form of the product and the C4a-hydroxyflavin. During the reduction of the enzyme-2,4-dihydroxybenzoate complex by NADPH with 2, 4-dihydroxybenzoate, a rapid transient increase in flavin absorbance is observed prior to hydride transfer from NADPH to FAD. This is direct evidence for movement of the flavin before reduction occurs.
在天然对羟基苯甲酸羟化酶的晶体结构中，丝氨酸212与对羟基苯甲酸的一个羧基氧原子之间的氢键距离为2.7埃。在本研究中，我们将第212位残基突变为丙氨酸，以研究丝氨酸氢键对酶功能的重要性。突变型和野生型（WT）酶与天然底物对羟基苯甲酸的比较表明，该残基有助于底物结合。该底物的解离常数比野生型高1个数量级，但催化过程在其他方面未发生变化。当使用替代底物2,4 - 二羟基苯甲酸时，会形成两种产物（2,3,4 - 三羟基苯甲酸和2,4,5 - 三羟基苯甲酸），这表明该底物可以以两种方向结合。动力学研究提供了证据，表明先前在野生型酶与该底物的氧化半反应中观察到的具有高消光系数的中间体是由产物的双烯酮形式和C4a - 羟基黄素共同作用形成的。在用2,4 - 二羟基苯甲酸通过NADPH还原酶 - 2,4 - 二羟基苯甲酸复合物的过程中，在氢化物从NADPH转移到FAD之前，观察到黄素吸光度迅速短暂增加。这是还原发生前黄素移动的直接证据。
