pH-Dependent Structural Changes in the Active Site of p-Hydroxybenzoate Hydroxylase Point to the Importance of Proton and Water Movements during Catalysis
详细信息 查看全文
- 作者：Domenico L. Gatti ; Barrie Entsch ; David P. Ballou ; and Martha L. Ludwig
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：January 16, 1996
- 年：1996
- 卷：35
- 期：2
- 页码：567 - 578
- 全文大小：836K
- 年卷期：v.35,no.2(January 16, 1996)
- ISSN：1520-4995
文摘
Deprotonation of p-hydroxybenzoate to the phenolate andreprotonation of the hydroxylateddienone intermediate to form the product are essential steps in thereaction catalyzed by p-hydroxybenzoatehydroxylase (PHBH). The mechanism by which protons are transferredin these reactions is not obvious,because the substrate bound in the active site is isolated fromsolvent. Structure analyses of wild-typeand mutant PHBH, with bound p-hydroxybenzoate orp-aminobenzoate, reveal a chain of protondonorsand acceptors (the hydroxyl groups of Tyr201 and Tyr385, and two watermolecules) that can connect thesubstrate 4-OH to His72, a surface residue. This chain couldprovide a pathway for proton transfer toand from the substrate. Using various combinations of pH andsubstrates, we show that in crystallinePHBH ionizable groups in the chain may rotate and change hydrogen-bondorientation. Molecular dynamicssimulations have been used to predict the preferred orientation ofhydrogen bonds in the chain as a functionof the ionization states of substrate and His72. The calculationssuggest that changes in the ionizationstate of the substrate could be associated with changes in orientationof the hydrogen bonds in the chain.Transfer of water between the chain of proton donors and thesolvent also appears to be an essential partof the mechanism that provides reversible transfer of protons duringthe hydroxylation reaction.