CAJ | 학술논문

γ-谷氨酰转肽酶(GGT;EC 2.3.2.2)催化的酰基转移反应可用于制备各种具有生理活性的谷氨酰化合物,对其开展酶活力不可逆抑制动力学研究对于阐明GGT的作用机制具有重要意义.今以化学抑制剂Woodward's Reagent K (WRK)与枯草芽孢杆菌NX-2产出的 GGT进行不可逆抑制反应,根据邹氏理论测得WRK对GGT不可逆抑制反应的微观速率常数ki为0.03015 s~(-1),WRK与酶结合常数KI为1.352 mmol·L~(-1).有抑制剂存在下GGT与供体γ-谷氨酰对硝基苯胺的亲和力常数K_m~*=3.245 mmol·L~(-1),GGT酰基化最大反应速度V_(max)~*=0.3771 mmol· (L·s)~(-1).通过对GGT的失活动力学分析得到,失活反应级数为1.313,说明在GGT活性部位至少有一个谷氨酸(或天冬氨酸)残基参与催化反应.
γ-곡안선전태매(GGT;EC 2.3.2.2)최화적선기전이반응가용우제비각충구유생리활성적곡안선화합물,대기개전매활력불가역억제동역학연구대우천명GGT적작용궤제구유중요의의.금이화학억제제Woodward's Reagent K (WRK)여고초아포간균NX-2산출적 GGT진행불가역억제반응,근거추씨이론측득WRK대GGT불가역억제반응적미관속솔상수ki위0.03015 s~(-1),WRK여매결합상수KI위1.352 mmol·L~(-1).유억제제존재하GGT여공체γ-곡안선대초기분알적친화력상수K_m~*=3.245 mmol·L~(-1),GGT선기화최대반응속도V_(max)~*=0.3771 mmol· (L·s)~(-1).통과대GGT적실활동역학분석득도,실활반응급수위1.313,설명재GGT활성부위지소유일개곡안산(혹천동안산)잔기삼여최화반응.
γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) can catalyze the cleavage compounds and the transfer of their γ-glutamyl groups to water or other amino acids and peptides. GGT can be used in the enzymatic synthesis of some glutamyl compounds with special physiological activities, and its inhibition is important for the investigation of its catalytic mechanism. The kinetic theory of the substrate reaction with irreversible inhibition of enzyme activity proposed previously by Tsou was applied for the study of the inactivation of GGT, and the irreversible inhibitor of Woodward's Reagent K (WRK) was used in this study to react with GGT from B. subtilis NX-2. As a result, the irreversible inhibition reaction rate of WRK to GGT,and the combination constant of WRK with enzyme were determined as k_i = 0.03015 s~(-1) and K_I =1.352 mmol·L~(-1), respectively. When the inhibitor presents, the Michaelis-Menten constant and the GGT maximum acylation reaction rate were found as K_m~*= 3.245 mmol·L~(-1) and V_(max)~*= 0.3771 mmol·(L·s)~(-1), respectively. Kinetic analysis reveals that the average order of the inactivation reaction is 1.313, and it indicates that there is at least one molecule of Glu/Asp residue exists in the active site of GGT, which plays the role of binding with the substrate during the catalytic reaction.