Donella-Deana A, Stone S R, Pinna L A
Dipartimento di Chimica Biologica, Università di Padova, Italy.
Eur J Biochem. 1991 Oct 15;201(2):501-5. doi: 10.1111/j.1432-1033.1991.tb16309.x.
Hirudin, the powerful anticoagulant agent of leech (Hirudo medicinalis) saliva, was readily phosphorylated by two spleen tyrosine protein kinases (TPK-IIB and TPK-III) at Tyr63 with Km values of 238 microM and 74 microM, respectively. The synthetic tridecapeptide DGDFEEIPEEYLQ, corresponding to the hirudin 53-65 C-terminal fragment, was phosphorylated even more efficiently than hirudin itself. Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin. The mutant in which all four glutamates were substituted, [Gln57,58,61,62]hirudin, was virtually not phosphorylated by either TPK-IIB and TPK-III. Substitution of Glu57 and Glu58 was less deleterious than substitution of the two glutamic acids adjacent to Tyr63: [Gln61,62]hirudin exhibited a 20-fold lower phosphorylation efficiency with TPK-IIB. With TPK-III, however, the Km value of [Gln61,62]hirudin was slightly lower, while the Vmax decreased sixfold. The substitution of Glu62 alone was also more detrimental with TPK-IIB than with TPK-III. The behaviour of a third spleen TPK, named lyn TPK-I and belonging to the src family, was markedly different in that it did not phosphorylate hirudin but exhibited significant activity towards [Gln57,58,61,62]hirudin. Taken together, these data confirm and extend with a protein substrate the results obtained with short model peptides which indicated the stringent substrate requirements of TPK-IIB (and of TPK-III to a lesser extent) for N-terminal acidic residues. In contrast, such residues are deleterious with lyn TPK-I. These observations also support the concept that tyrosine protein kinases recognize specificity determinants situated in the vicinity of the target residue rather than requiring higher-order structural features.
水蛭（医用水蛭）唾液中的强效抗凝剂水蛭素很容易被两种脾脏酪氨酸蛋白激酶（TPK-IIB和TPK-III）在Tyr63位点磷酸化，其Km值分别为238微摩尔和74微摩尔。与水蛭素53 - 65 C末端片段相对应的合成十三肽DGDFEEIPEEYLQ的磷酸化效率甚至比水蛭素本身还要高。四种水蛭素突变体，其中57、58、61和62位的一个或多个谷氨酸被谷氨酰胺取代，它们作为底物的效果比水蛭素要差。所有四个谷氨酸都被取代的突变体[Gln57,58,61,62]水蛭素几乎不被TPK-IIB和TPK-III磷酸化。Glu57和Glu58的取代比Tyr63相邻的两个谷氨酸的取代危害小：[Gln61,62]水蛭素与TPK-IIB的磷酸化效率低20倍。然而，对于TPK-III，[Gln61,62]水蛭素的Km值略低，而Vmax降低了六倍。单独取代Glu62对TPK-IIB的危害也比对TPK-III的危害更大。第三种脾脏TPK，名为lyn TPK-I，属于src家族，其行为明显不同，它不使水蛭素磷酸化，但对[Gln57,58,61,62]水蛭素有显著活性。综上所述，这些数据用一种蛋白质底物证实并扩展了用短模型肽获得的结果，这些结果表明TPK-IIB（以及在较小程度上TPK-III）对N末端酸性残基有严格的底物要求。相比之下，这些残基对lyn TPK-I是有害的。这些观察结果也支持这样一种概念，即酪氨酸蛋白激酶识别位于靶残基附近的特异性决定因素，而不是需要更高阶的结构特征。
