Mutant rat trypsin selectively cleaves tyrosyl peptide bonds.
A double mutant of rat trypsinogen (Asp189Ser, DeltaAsp223) was constructed by site-directed mutagenesis. The recombinant protein was produced in Escherichia coli under the control of a periplasmic expression vector. The purified and enterokinase-activated enzyme was characterized by synthetic fluorogenic tetrapeptide and natural polypeptide substrates and by a recently developed method. In case of this latter method the specificity profile of the enzyme was examined by simultaneous digestion of a mixture of oligopeptide substrates each differing only at the P(1) site residue, and the results were analyzed by high-performance liquid chromatography. All these assays unanimously demonstrated that the recombinant proteinase lacks trypsin-like activity but acquired a rather unique selectivity: it preferentially hydrolyses peptide bonds C-terminal to tyrosyl residues. This narrow specificity should be useful in peptide-analytical applications such as sequence-specific fragmentation of large proteins prior to sequencing.
Department of Biochemistry, Eötvös Loránd University, Pázmány St. 1/c, 1117 Budapest, Hungary.
Gábor
G
Pál
András
A
Patthy
József
J
Antal
László
L
Gráf
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014362:Trypsinogen / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014362:Trypsinogen; D014357:Trypsin