利用定點飽和突變研究抹香鯨肌紅蛋白中Ile-107位置對其過氧化能力之影響

Abstract

肌紅蛋白(Myoglobin)為一種血基質蛋白(heme-protein)，在脊椎動物體內具有儲存及攜帶氧氣的功能。本論文的研究主要是將不具酵素活性的肌紅蛋白突變成具有過氧化酵素(peroxidase)活性的功能。將mb基因殖入pET28a(+)表現質體中，利用定點突變及定點飽和突變技術改變His-64、Val-68、Ile-107這三個推測可改變活性的胺基酸。利用IPTG誘導蛋白質的大量表現以形成包涵體(inclusion body)，接著以guanidine hydrochloride使包涵體變性再復性後，利用DEAE管柱純化，可以得到純度高達90%的脫輔基肌紅蛋白(apo-myoglobin)，利用蛋白質輔基重組實驗可將血基質(heme)包入突變的脫輔基肌紅蛋白中。與過氧化氫和2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)反應後可以利用UV/VIS光譜儀偵測其一個電子傳遞的過氧化酵素活性。比較文獻的雙重突變H64D/V68L Mb以及利用定點飽和突變技術改變Ile-107的三重突變，在經由動力學參數測量後發現H64D/V68L/I107M Mb在與過氧化氫形成Compound I的效率可提高30%，與ABTS的一個電子傳遞效率則提高60%。另外也發現將Ile-107置換成體積較小的Ala及Val對形成Compound I的效率影響不大，但是卻可以提高一個電子傳遞效率47%及36%。因此認為Ile-107可能藉由立體效應去影響酵素與受質的一個電子傳遞效率。未來本實驗室會將各種不同金屬的紫質包入突變的脫輔基肌紅蛋白中，並研究其特性以期能應用並開發新型的染料敏化生物性太陽能電池(dye-sensitized biosolar cell)。

Myoglobin is a heme-protein, functioning as oxygen storage/carrier in vertebrates. In this study, the nonenzymatic myoglobin was functionally converted into a heme enzyme with peroxidase activity. Three amino acid residues, His-64/Val-68/Ile-107, located on the putative active site cavity, were subjected to site-directed mutagenesis and cloned into a pET-28a(+) expression vector. Following IPTG induction, the cell cultures were harvested and subjected to protein purification. After guanidine hydrochloride disruption and renaturation, the soluble apo-myoglobin was purified to homogeneity by DEAE chromatography. The heme molecule was reconstituted into these renatured apo-myoglobin mutants. A well established peroxidase activity with one-electron oxidation of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was observed by the UV/VIS spectrophotometer from the myoglobin mutants, demonstrating its capability in electron transfer reaction. Among these mutations, as compared with the original MbH64D/V68L double mutant, MbH64D/V68L/I107M triple mutant exhibited a 30% activity increase in the compound I formation, whereas, a 60% activity increase in the one-electron oxidation was observed. Alternativily, the MbH64D/V68L/I107A or MbH64D/V68L/I107V showed the similar compound I formation rate, but with the one-electron oxidation rate of 47% or 36% increment respectively. These results indicated that the Ile-107 position may influence the one-electron oxidation of substrates via steric effect after the compound I formation. In the future, various metalloporphyrins with different metal ions will be reconstituted into the apo-myoglobin mutants to investigate their potentials as novel dye-sensitizers for biosolar cell application.