Ramaswamy S G
J Biol Chem. 1984 Jan 10;259(1):249-54.
Hydroxyproline 2-epimerase of Pseudomonas putida was purified to homogeneity by an improved procedure. The native enzyme consists of two probably identical subunits. Alkylation of the active site with labeled reagents resulted in the loss of 80-85% of the activity but the incorporation of only one alkyl group even though the active site contains a Cys residue from each of the two subunits. This result suggests that the enzyme shows half-site reactivity. The labeled enzyme was further subjected to exhaustive alkylation with unlabeled iodoacetate, permitting tryptic hydrolysis and isolation of an active site peptide in 30% yield. The specific radioactivity of the peptide was consistent with the first result, that only 1 mol of alkyl group was initially incorporated into active site. The active site peptide (14 residues) was sequenced and found to possess homology with the clostridial proline racemase.
通过改进的方法，将恶臭假单胞菌的羟脯氨酸2-差向异构酶纯化至同质。天然酶由两个可能相同的亚基组成。用标记试剂对活性位点进行烷基化导致80-85%的活性丧失，但仅掺入一个烷基，尽管活性位点含有来自两个亚基中每个亚基的一个半胱氨酸残基。该结果表明该酶表现出半位点反应性。将标记的酶进一步用未标记的碘乙酸进行彻底烷基化，进行胰蛋白酶水解并以30%的产率分离出活性位点肽段。该肽段的比放射性与第一个结果一致，即最初仅1摩尔烷基被掺入活性位点。对活性位点肽段（14个残基）进行测序，发现其与梭菌脯氨酸消旋酶具有同源性。
