进化orotidine酶活动的5 '一磷酸脱羧酶suprafamily:机械的证据一个质子中继系统的活性部位3-keto-L-gulonate 6-phosphate脱羧酶。
文章的细节
-
引用
复制到剪贴板 -
紫杉WS、明智的EL Rayment我Gerlt农协
进化orotidine酶活动的5 '一磷酸脱羧酶suprafamily:机械的证据一个质子中继系统的活性部位3-keto-L-gulonate 6-phosphate脱羧酶。
生物化学。2004年6月1日,43 (21):6427 - 37。
- PubMed ID
-
15157077 (在PubMed]
- 文摘
-
3-Keto-L-gulonate 6-phosphate脱羧酶(KGPDC)和orotidine 5 '一磷酸脱羧酶(OMPDC)同源酶分享(β/α)(8)倍但催化不同反应[智慧,E。紫杉,w·S。,巴比特,p . C。Gerlt, j。,Rayment i(2002)生物化学41岁,3861 - 3869]。KGPDC催化镁(2 +)端依赖脱3-keto-L-gulonate 6-phosphate,异化的途径的一个中间L-ascorbate由大肠杆菌利用k - 12(紫杉,w·S。Gerlt, j·a·j . Bacteriol(2002)。184年,302 - 306]。OMPDC催化金属ion-independent反应可能收益没有乙烯阴离子中间(Appleby, t . C。Kinsland C。贝格利,T。,Ealick s e (2000) Proc。国家的。学会科学。美国97年,2005 - 2010],虽然机械的细节是不确定的。结束一个活跃的站点赖氨酸位于第三beta-strand OMPDC提出了一般的酸,送一个solvent-derived质子人民运动联盟产品;KGPDC包含一个同源赖氨酸残基的活性部位(Lys64)。 Herein, we report investigations of the KGPDC-catalyzed reaction that are consistent with a mechanism involving a Mg(2+)-stabilized cis-enediolate intermediate [Wise, E. L., Yew, W. S., Gerlt, J. A., and Rayment, I. (2003) Biochemistry 42, 12133-12142] and implicate waters proximal to His136 and Arg139, both located at the end of the sixth beta-strand, as the general acids that deliver a solvent-derived proton to the intermediate to form the L-xylulose 5-phosphate product. On the basis of our mechanistic investigations, Lys64 stabilizes the cis-enediolate intermediate by forming hydrogen bonds to both O1 and O2 of the intermediate. Thus, although the active sites of OMPDC and KGPDC contain a conserved Lys at the end of the third beta-strand, their roles in catalysis are not conserved. Furthermore, a conserved Asp at the end of the third beta-strand in OMPDC participates in a hydrogen-bonded network that positions the acidic Lys residue; in the active site of KGPDC, the homologous Asp67 participates in stabilization of the enediolate intermediate and enforces a cis geometry. We conclude that the conserved active site residues perform different functions in the OMPDC- and KGPDC-catalyzed reactions, so the mechanisms of their reactions are completely distinct. This study further highlights the opportunistic nature of divergent evolution in conscripting the active site of a progenitor to catalyze a mechanistically distinct reaction.