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首页> 外文期刊>Biochemistry >Analysis of tissue plasminogen activator specificity using peptidyl fluorogenic substrates.
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Analysis of tissue plasminogen activator specificity using peptidyl fluorogenic substrates.

机译：使用肽基荧光底物分析组织纤溶酶原激活剂特异性。

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A series of 54 fluorogenic substrates have been synthesized and evaluated for tissue-type plasminogen activator (tPA) hydrolysis in an attempt to create efficient sensitive substrates for tPA and to investigate substrate structure-efficiency correlations. All substrates contain the 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P1 position, various amino acids in the P2 and P3 positions, and various substituents in the sulfonamide moiety of the leaving group (P' position). The majority of substrates have relatively low K(M) values (< 100 microM), reaching as low as 2.6 microM, and reasonably high k(cat) values (up to 3.6 s(-1)). These substrates have higher affinity, higher hydrolysis rates, and higher efficiency for two-chain tPA than for the single-chain form of this enzyme. Analysis of the P3 structure influence on substrate efficiency demonstrates that compounds which contain D-isomers of N-blocked bulky amino acids, such as Phe, Leu, and Val, in this position are more efficient for tPA than substrates with N-unblocked small amino acids (Ser or Pro) in the P3 position. The second-order rate constants and k(cat) values for substrate hydrolysis increase with decreases in the P2 amino acid hydrophobicity in the following manner: Leu < Val and Gly < Ser < Pro. Substrates which contain an ANSN leaving group had a higher affinity for tPA than substrates with p-nitroaniline or 7-amino-4-methylcoumarin leaving groups. Analyses of substrate hydrolysis dependence on the substrate P' structure show that the k(cat) and the second-order rate constants increased with an increase in the size of monoalkyl substituent in the sulfonamide moiety, whereas substrates which contain either glycine methyl ester or a dialkyl group displayed the lowest efficiency for tPA. The substrate Boc-(p-F)Phe-Pro-Arg-ANSNHC2H5 allowed quantitation of tPA at a concentration as low as 1 pM, a concentration significantly lower than the plasma concentration of this protein. Evaluation of the activation of single-chain tPA by factor Xa demonstrates that prothrombinase is approximately 3-fold more efficient in activating sc-tPA than factor Xa alone, increasing the initial rate of activation from 0.0055 nM/s per 1 nM of factor Xa to 0.017 nM/s per 1 nM.
机译：合成了一系列54种荧光底物，并评估了其对组织型纤溶酶原激活剂（tPA）的水解作用，以尝试创建有效的tPA敏感底物并研究底物结构与效率的相关性。所有底物均包含6-氨基-1-萘磺酰胺（ANSN）离去基团，P1位置的Arg，P2和P3位置的各种氨基酸以及离去基团的磺酰胺部分（P'位置）的各种取代基。大多数底物具有相对较低的K（M）值（<100 microM），低至2.6 microM和相当高的k（cat）值（高达3.6 s（-1））。这些底物对于双链tPA具有比该酶的单链形式更高的亲和力，更高的水解速率和更高的效率。 P3结构对底物效率的影响分析表明，在此位置上包含N封闭的大氨基酸D-异构体（例如Phe，Leu和Val）的化合物对tPA的作用比N封闭的小氨基的底物更有效P3位置的酸（Ser或Pro）。底物水解的二级速率常数和k（cat）值随着P2氨基酸疏水性的降低而增加，其方式如下：Leu

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