Prolyl 4-hydroxylase converts collagen prolines to 4-hydroxyprolines

Collagen was for many years considered the only source of 4-hydroxyproline (4-Hyp) in animals. Though it is now known that other proteins such as C1q and elastin also contain 4-Hyp, collagen is by far the major source (Adams & Frank 1980). 4-Hyp is required for collagen stability at physiological temperatures. The abundance of Hyp in animal proteins is ~4%, making it more abundant than the amino-acids Cys, Gln, His, Met, Phe, Trp and Tyr (McCaldon & Argos 1988). In collagen Hyp abundance is much higher at ~38% (Ramshaw et al. 1998). Full collagen proline hydroxylation significantly raises the melting temperature (Tm) by stabilizing the collagen triple helix (Berg & Prockop 1973a), a process that has been studied extensively using synthetic collagen peptides (Sakakibara et al. 1973, Holmgren et al. 1998) and is well understood at the structural level (Shoulders & Raines 2009). Collagen 4-Hyp content is relatively stable, with small differences between collagen types. Collagen type I has approximately 1 4-Hyp for every 10 residues, roughly 50% of available prolines (Kivirikko et al. 1992). Conversion of Pro to (2S,4R)-4-hydroxyproline (4-Hyp) is the most prevalent posttranslational modification in humans, catalyzed by prolyl 4-hydroxylase (P4H). Mammalian prolyl 4-hydroxylase is an alpha2 beta2 tetramer (Berg & Prockop 1973b). The 59-kDa alpha subunit contains the substrate-binding domain and the enzymic active site (Helaakoski et al. 1989). Humans and most other vertebrates have three isoforms of the alpha subunit, isoform alpha-1 is the most prevalent. The pair of alpha subunits can be any of the three isoforms (Gorres & Raines 2010). The 55-kDa beta subunit is protein disulphide isomerase (PDI), which has additional functions in collagen formation. As part of P4H it retains the tetramer in the ER lumen and maintains the otherwise insoluble alpha subunit in an active form (Vuori et al. 1992, Nietfeld & Kemp 1981). P4H is a member of the non-heme iron(II), alpha-ketoglutarate-dependent dioxygenase family. Molecular oxygen (O2), 2-oxoglutarate (alpha-ketoglutarate) and iron(II) are required for its activity (Hutton & Udenfriend 1966). During the reaction, alpha-ketoglutarate is oxidatively decarboxylated producing succinate and CO2 (Rhoads & Udenfriend 1968, Gorres & Raines 2010). Ascorbate is required as a cofactor but not consumed (Kivirikko et al 1989). The minimum substrate required for hydroxylation is an Xaa-Pro-Gly tripeptide, with Pro preferred in the Xaa position, though hydroxylation can occur at lower rates with a variety of residues at this position (Kivirikko et al. 1972). A number of other peptides, notably elastin, are substrates for P4H (Bhatnagar 1978).<br><br>For brevity, all forms of collagen propeptide are shown as having 3X 4-Hyp residues following the action of P4H.

胶原蛋白长期以来被视为动物体内4-羟基脯氨酸（4-Hyp）的唯一来源。尽管现已知晓C1q和弹性蛋白等其他蛋白质亦含有4-Hyp，但胶原蛋白依然是其主要的来源（Adams & Frank 1980）。4-Hyp对于胶原蛋白在生理温度下的稳定性至关重要。动物蛋白中Hyp的含量约为4%，使其比半胱氨酸、谷氨酰胺、组氨酸、甲硫氨酸、苯丙氨酸、色氨酸和酪氨酸等氨基酸更为丰富（McCaldon & Argos 1988）。在胶原蛋白中，Hyp的含量显著更高，约为38%（Ramshaw et al. 1998）。全胶原蛋白的脯氨酸羟基化通过稳定胶原蛋白的三股螺旋结构（Berg & Prockop 1973a），显著提高了其熔点（Tm），这一过程已通过合成胶原蛋白肽（Sakakibara et al. 1973, Holmgren et al. 1998）进行了广泛研究，并在结构层面上得到了充分理解（Shoulders & Raines 2009）。胶原蛋白中的4-Hyp含量相对稳定，不同类型胶原蛋白之间的差异微小。胶原蛋白I型中，每10个残基中大约含有1个4-Hyp，约占可用脯氨酸的50%（Kivirikko et al. 1992）。脯氨酸转化为（2S,4R）-4-羟基脯氨酸（4-Hyp）是人类中最常见的翻译后修饰，由脯氨酸4-羟基化酶（P4H）催化。哺乳动物的P4H是一种α2β2四聚体（Berg & Prockop 1973b）。59kDa的α亚基含有底物结合域和酶活性位点（Helaakoski et al. 1989）。人类和大多数其他脊椎动物具有三种α亚基的同型异构体，其中同型异构体α-1是最为普遍的。成对的α亚基可以是三种同型异构体中的任何一种（Gorres & Raines 2010）。55kDa的β亚基是蛋白质二硫键异构酶（PDI），在胶原蛋白形成中具有额外功能。作为P4H的一部分，它保留了内质网腔中的四聚体，并维持了原本不溶的α亚基处于活性状态（Vuori et al. 1992, Nietfeld & Kemp 1981）。P4H属于非血红素铁(II)、α-酮戊二酸依赖性二氧合酶家族。分子氧（O2）、2-氧戊二酸（α-酮戊二酸）和铁(II)是其实际所必需的（Hutton & Udenfriend 1966）。在反应过程中，α-酮戊二酸被氧化脱羧产生琥珀酸和二氧化碳（Rhoads & Udenfriend 1968, Gorres & Raines 2010）。抗坏血酸作为辅因子是必需的，但不会被消耗（Kivirikko et al 1989）。羟基化的最小底物是Xaa-Pro-Gly三肽，其中Pro在Xaa位置最为理想，尽管在此位置使用其他各种残基羟基化的速率较低（Kivirikko et al. 1972）。P4H的底物还包括多种其他肽，尤其是弹性蛋白（Bhatnagar 1978）。为了简洁起见，在P4H作用后，所有形式的胶原蛋白前肽均显示含有3个4-Hyp残基。