转酰胺酶动力学。硫醇酯与胺的酶促反应中酰胺的形成。

Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.

作者信息

Stenberg P, Curtis C G, Wing D, Tong Y S, Credo R B, Gray A, Lorand L

出版信息

Biochem J. 1975 Apr;147(1):155-63.

PMID:239698

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1165385/

Abstract

Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.

摘要

β-苯丙酰硫代胆碱和N-(5-氨基戊基)-5-二甲基氨基萘-1-磺酰胺（丹磺酰尸胺）用作一对用于转酰胺酶的水溶性（pH7.5）模型底物。酰胺形成可通过荧光测量直接跟踪，方法是监测水溶性偶联产物N-(β-苯丙酰基)丹磺酰尸胺持续萃取到正庚烷中的情况。通过此程序，在25℃下研究了人血浆（纤维蛋白溶解酶、凝血酶和Ca2+激活的血液凝固因子XII）和豚鼠肝脏（肝脏转谷氨酰胺酶）中谷氨酰胺-赖氨酸内切γ-谷氨酰胺转移酶的稳态动力学。2. 以β-苯丙酰硫代胆碱作为变化的底物，不同浓度丹磺酰尸胺的Lineweaver-Burk图在横轴上截距，表明酰基酶的形成是限速步骤。3. 根据活性位点的功能正常性，分别获得血浆和肝脏γ-谷氨酰胺转移酶的kcat.值为1.8 s(-1)和0.9 s(-1)。这两种酶对第一种底物β-苯丙酰硫代胆碱具有相同的亲和力，Ka为4×10(-4) M。4. 第二种底物丹磺酰尸胺的利用，肝脏酶似乎效率高一个数量级。5. N-(5-氨基-3-硫代戊基)-5-二甲基氨基萘-1-磺酰胺（丹磺酰硫代尸胺）可在荧光动力学系统中代替丹磺酰尸胺使用。6. 还评估了非荧光胺底物组胺的竞争性抑制作用。