Direct and continuous assay for prolyl 4-hydroxylase
Direct and continuous assay for prolyl 4-hydroxylase
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DOI：
10.1016/j.ab.2008.11.046
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发表时间：
2009-03-15
影响因子：
2.9
通讯作者：
Raines, Ronald T.
中科院分区：
文献类型：
--
作者：
Gorres, Kelly L.;Raines, Ronald T.
脯氨酰4-羟化酶（P4 H）是一种非血红素铁双加氧酶，催化原胶原链中（2S）-脯氨酸（Pro）残基的翻译后羟化。所得的（2S，4 R）-4-羟脯氨酸（Hyp）残基对于胶原三螺旋的折叠、分泌和稳定性是必需的。P4 H使用α-酮戊二酸和O-2作为共底物，并形成琥珀酸和CO2以及Hyp。本文描述的是连续且直接检测含脯氨酸底物的周转的第一种P4 H测定。本试验基于（2S，4S）-4-氟脯氨酸（flp），一种通过P4 H转化为（2S）-4-酮脯氨酸（Kep）和无机氟化物的脯氨酸类似物。通过氟离子选择性电极检测氟离子，从而通过P4 H进行周转。使用该测定法，测定了人P4 H催化的含flp肽的周转的稳态动力学参数，发现其与使用基于不连续HPLC的测定法获得的参数相当。此外，该测定可用于表征P4 H变体，如通过D414 A P4 H和野生型酶的催化作用的比较所证明的。最后，通过在2，4-吡啶二羧酸（α-酮戊二酸的类似物）存在下的催化分析，验证了使用该测定来鉴定P4 H的小分子抑制剂。因此，本文所述的测定法可以促进这种必需酶的生物化学分析。(C)2008年爱思唯尔公司All rights reserved.
Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses alpha-ketoglutarate and O-2 as cosubstrates, and forms succinate and CO2 as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing Substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to Characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of alpha-ketoglutarate. Thus, the assay described herein Could facilitate biochemical analyses of this essential enzyme. (C) 2008 Elsevier Inc. All rights reserved.