Specificity determinants for tyrosine protein kinase. A study with recombinant hirudin mutants.
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 Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin.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.
Dipartimento di Chimica Biologica, Università di Padova, Italy.
A
A
Donella-Deana
S R
SR
Stone
L A
LA
Pinna
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006629:Hirudins / Q000235:genetics / Q000378:metabolism*; D007865:Leeches; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases