- 學位論文
以定位突變探討Oxa-66乙內醯胺分解酶129位置對受質imipenem酵素動力學與酵素羧化的影響
Site-directed mutagenesis of position I129 in Oxa-66 β-lactamase: Effect of aliphatic side chain on substrate imipenem enzyme kinetics and enzyme carboxylation
摘要
Oxacillinase乙內醯胺分解酶，簡稱OXA，被歸類為Class D b-lactamases，會分解carbapenem類抗生素Impenem (IMP)、Meropenem等。由於Oxacillinase絕大部分存在於 Acinetobacter baumannii (AB)菌，因而造成近年來AB菌抗藥性的急遽上升。目前AB菌的OXAs主要有OXA-23 like、OXA-40/24 like、OXA-51 like以及OXA-58 like 4個亞群，其中OXA-66屬於OXA-51 like，是AB菌與生俱有的基因。從已解開的OXA-24晶體結構得知，受質會從通道狀的入口進入活化中心。OXA-66與OXA-24為同源蛋白，其相對應胺基酸位置129 Ile就位於通道狀的入口處，並在空間分布上與活化中心S80及K83相鄰。K83羧化與否會影響Class D b-lactamases酵素與受質的去醯化能力。過去本研究室發現當OXA-66 I129點突變為Leu時，其分解IMP的活性增加3.5倍。因此，我們推測OXA-66 I129除了會影響酵素與受質的親和力外也有可能會影響K83的羧化能力。 為了釐清OXA-66 I129所扮演的角色 ，本實驗室以基因選殖技術與點突變技術取得OXA-66及其位置I129突變為疏水性胺基酸L、V、A及非疏水性G的重組蛋白。並以分光光度儀觀察這些重組蛋白對於受質IMP的活性，並以酵素動力理論分析。最後利用Swiss PDB Viewer模擬空間關係。 結果發現Km (uM) (L ≑ I < V < A)分別為13.3 (L)、13.2 (I)、10.3(V)、9.2 (A)。配合空間模擬，我們認為除了I129G外，其它蛋白的通道狀入口均隨著胺基酸分子量的變小而使得入口處變寬，因而增加酵素與受質IMP的親和力；而Vmax相對值比較則分別為 100% (I)、381% (L)、115% (V)、155% (A)、124% (G)，I129L並不因Km變大而使酵素活性變小，因此推測129位置的胺基酸有可能也會影響酵素的催化能力。 更進一步釐清OXA-66 129位置的胺基酸是否對K83羧化能力的影響，實驗中加入NaHCO3，並進行活性測量與分析。結果發現重組蛋白對CO2的Km (mM) (L < I < G)分別為2.89 (L)、7.82 (I) 、9.06 (G)，推論I129L使得K83對CO2親和力較大，I次之，I129G最小。從空間模擬圖可以得知，129L與K83的距離最近為3.14 Å，I次之3.53 Å，G最遠3.87 Å。由於L的疏水特性且又最靠近K83，使得K83有較小的pKa值，較容易形成羧化態。因此我們推論OXA-66 129位置會影響K83周圍環境的疏水性程度，進而影響K83的的羧化能力。 綜合以上結論，我們認為在OXA-66的129位置不僅影響受質與酵素的親和力外，更重要是增強K83周圍的疏水環境，促使K83得以有較強的羧化能力，以進行受質的水解反應。
並列摘要
Oxacillinas (OXA) is a class D b-lactamase, represents an emerging antibiotic resistance mechanism encountered among the most opportunistic Gram-negative bacterial pathogens, especially Acinetobacter baumannii. At present, A. baumannii OXAs mainly has four groups: OXA-23 like group, OXA-40/24 like group, OXA-51 like group and OXA-58 like group. Among them, OXA-66, group of OXA-51 like, is naturally occurring in A. baumannii. The recently solved crystal structure of OXA-24 suggests the tunnel-like entrance to the active site. Comparison of the homology model of OXA-66, the position I129 of OXA-66 is on the tunnel-like entrance and interact with active site S80 and N-carboxylated K83. Our laboratory constructed serveral mutants at position I129 with aliphatic side chain L/V/A/G by site-directed mutagensis, in order to investigate how variants affect the ability of hydrolyzing substrate impenem and N-carboxylated K83 by enzyme kinetics analysis. Finally, we simulated interactions between I129 with N-carboxylated K83 by software Swiss pdb viewer . Our data shows (i) Km (uM) ranked 13.3 (L), 13.2 (I), 10.3(V), 9.2 (A), 22.6 (G). It estimated the IMP affinity decreased as molecular size of position 129 side chain, but Gly. (ii) relative Vmax ranked 100% (I)、381% (L)、115% (V)、155% (A)、124% (G). It showed position 129 affects the reaction velocity. Furthermore, we measured CO2 affinity: (iii) Km (mM) ranked 9.06 (I), 2.89 (L), 7.82 (G) is tending with distance between side chain of position 129 and N-carboxylated K83. We suggest the side chain at position 129 affect CO2 binding to K83 by creating hydrophobic environment. Our data suggest that position 129 of OXA-66 not only affects the affinity with substrate IMP, but also affects CO2 binding to K83 by creating hydrophobic environment.