以定位突變探討Oxa-66乙內醯胺分解酶129位置對受質meropenem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 meropenem enzyme kinetics and enzyme carboxylation

10.6834/CSMU.2013.00196

吳宗翰

乙內醯胺分解酶AB菌 ； oxacillinase ； Class D beta-lactamases ； Acinetobacter baumannii

中山醫學大學生化暨生物科技研究所學位論文

2013年

碩士

陳凌雲

繁體中文

臨床上使用carbapenem類抗生素Imipenem、Meropenem等對付具有Oxacillinase的感染性菌株。Acinetobacter baumannii的OXA-66屬於OXA-51 like，OXA-66胺基酸位置129 Ile位於受質與酵素催化的空間，在空間分布上與活化中心S80及K83相鄰。文獻指出129位置的疏水性胺基酸會降低K83的pKa值，使-NH2釋放質子接受CO2。K83羧化程度會影響Class D β-lactamases水解β-lactam的能力。過去本研究室發現當OXA-66 I129點突變為Leu時，分解meropenem的活性增加5.5倍。推測OXA-66 I129除了影響酵素水解受質的催化能力外也有影響K83的羧化能力。為了釐清OXA-66 I129所扮演的角色，本實驗以基因選殖與點突變取得OXA-66及其位置I129突變為疏水性胺基酸L、V、A、G的重組蛋白。觀察這些重組蛋白對於受質MER的活性，以酵素動力學理論分析。 結果發現Km ( μM )由小到大分別為59.7 ( L )、74.7 ( A )、87.9( I )、98.9( V )， I129L酵素對受質的親和力最佳，I129V較弱。推論129位置胺基酸的側鏈結構影響到酵素與受質的親和力；而Vmax相對值比較由大到小為224% ( L )、104% ( A )、100% ( I )、80.14% ( V )，可以觀察到I129L上升倍數最高，I129V最低，推測129位置胺基酸的改變，使酵素對受質的親和力影響到水解的能力。 在I129G的突變株重組蛋白對於受質meropenem的活性實驗中發現有Bi-phasic的現象，原因可能是129位置影響到K83羧化能力。 為了釐清OXA-66 129位置的胺基酸是否影響K83羧化能力，加入NaHCO3，並進行活性測量與分析，結果wild type活性上升91倍，I129L上升30倍，最後wild type與I129L酵素水解受質的活性幾乎達到一致，推測Lys的carboxylation對酵素催化受質的影響大於胺基酸側鏈造成的空間影響。 I129G水解受質meropenem時添加足量CO2發現原先的bi-phase轉變為mono-phase，可能因為屬於弱疏水性質的Glysine缺乏協助K83進行羧化。因此我們製備了I129D的重組蛋白，發現加入足量的CO2也無法使得OXA66-I129D對於meropenem有任何活性。因此推論OXA66 酵素水解受質meropenem的活性仰賴於129位置胺基酸基團的疏水能力。 為了驗證位置129疏水性基團對於Lys83的carboxylation的影響，進行酵素動力學分析 CO2對酵素水解受質merpenem的反應，顯示Km ( mM )由小到大分別為L(4.24)、I(5.62)、G(31.09)，推論129位置胺基酸基團的疏水能力會影響酵素水解受質過程中對於CO2的親和力，在未加入CO2時酵素對受質的酵素動力學實驗中，L的Km小於I，加入CO2對酵素化受質的酵素動力學實驗結果亦顯示L的Km小於I。Vmax值由分別為I(100%)、L(104%)、G(16%)，由於I、L的Vmax值沒有明顯差異，可能Lys83的carboxylation影響遠超過疏水性胺基酸側鏈所造成的空間差異，由以上結果推論129位置的疏水性胺基酸會影響Lys83進行carboxylation進而影響酵素催化受質的活性

Clinical use of carbapenem antibiotics Imipenem, Meropenem, etc. to deal with Oxacillinase infectious isolates. Acinetobacter baumannii bacteria OXA-66 belongs to OXA-51 like, are found in some Acinetobacter baumannii bacteria genes. OXA-66 amino acid position 129 Ile substrates and enzyme catalysis in space and in the spatial distribution and activation center Ser80 and Lys83 adjacent. 129 locations literature that will reduce the hydrophobic residues Lys83 the pKa value so that the release of a proton acceptor -NH2 for CO2. Lys83 carboxylation extent will affect the Class D beta-lactamases hydrolyze beta-lactam capabilities. Past of our laboratory found that when OXA-66 I129 point was mutated to Leu, the decomposition of the MER activity increased 5.5-fold. Speculate OXA-66 I129 addition to affecting the enzymatic hydrolysis of substrate also affect the catalytic capabilities of carboxylation capacity K83. OXA-66 I129 in order to clarify the role of the experiment, gene cloning and mutation to obtain OXA-66 and its location I129 mutated to hydrophobic amino acids L, V, A, G recombinant proteins. Observation of these recombinant proteins for substrates MER activity, an enzyme kinetic theory analysis. The result showed that Km in order were 59.7 (L), 74.7 (A), 87.9 (I), 98.9 (V), I129L observed affinity of the enzyme for the best substrates, I129V weak. Therefore Corollary 129 amino acid side chain structure position affects enzymes and substrates affinity; while descending Vmax relative value compared to 224% (L), 104% (A), 100% (I), 80.14% (V), can be observed up to a multiple of the highest I129L, but I129V be lower, suggesting that changes in amino acid position 129 of the enzyme affinity substrates affect the enzymatic hydrolysis catalyzed by a qualitative capability. In I129G mutant recombinant protein substrates meropenem for active experiments found that Bi-phasic phenomenon may be due to the 129 position affects the positioning of acetylated water molecules, or affect Lys83 carboxylation capacity. In order to clarify the position of OXA-66 129 amino acids would not affect Lys83 carboxylation capacity, adding NaHCO3, and conduct active measurements and analysis, the results wild type activity was increased 91-fold, I129L increased 30-fold, and the wild type enzyme hydrolysis of substrates with I129L activity almost identical, suggesting that the carboxylation Lys substrates for the enzyme catalytic amino acid side chain is greater than the impact caused by the spatial impact. I129G meropenem hydrolysis of substrate added when a sufficient amount of CO2 that the original bi-phase shift for the mono-phase, probably because of a weak hydrophobic nature Glysine assist Lys83 be carboxylase deficiency. Therefore, we prepared I129D recombinant protein, discovered that adding a sufficient amount of CO2 that Oxa66-I129D can not have any activity for meropenem. Therefore, by inference OXA66 enzymatic hydrolysis of the active substance meropenem relies on 129 position amino group of hydrophobic capability. To verify the position 129 for a hydrophobic group of Lys83 of carboxylation, for analysis of enzyme kinetics of enzymatic hydrolysis of substrate merpenem CO2 reaction shown Km (mM) Ascending were I (3.64), L (5.55) , V (6.14), A (9.97), G (35.93), Corollary 129 position amino group of hydrophobic ability to influence the course of the enzymatic hydrolysis of substrate affinity for CO2, CO2 is not added when the enzymes are affected by a qualitative enzyme kinetics experiments, L Km less than I, CO2 substrates for enzymes of the enzyme kinetic experiments also showed that L is smaller than the Km I. Vmax of the respectively I (100%), L (104%), G (16%), as I, L the Vmax value is not significantly different, as I, L, V, A are of hydrophobic Amino Acid, so the Vmax and no significant difference in comparison, the above results suggest that the hydrophobic amino acid position 129 affects Lys83 for carboxylation catalyst substrates thereby affecting the activity of the enzyme, and the better the hydrophobic amino acids groups such as I and L such that the enzymatic hydrolysis of substrate activity of meropenem slightly.