Alteration of the enzymatic specificity of human angiogenin by site-directed mutagenesis.
The molecular basis for the enzymatic specificity of human angiogenin has been investigated by site-directed mutagenesis of Thr-44, Glu-108, and Ser-118--residues corresponding to those thought to be involved in substrate base recognition in the homologous protein, RNase A. Mutations of Thr-44 to Ala, His, and Asp affect both activity and specificity dramatically. The Ala and His replacements decrease activity toward tRNA by factors of 25 and 40, respectively, and reduce cleavage of cytidylyl more than uridylyl dinucleotides. Substitution by Asp does not influence the rate of tRNA and rRNA degradation but alters specificity even more markedly than the other mutations: T44D-angiogenin has 17-40-fold decreased activity toward CpN' dinucleotides and 1.3-1.9-fold increased activity toward UpN', resulting in an inverted order of preference (U > C) compared to native angiogenin. Mutations of Glu-108 to Lys and Gln change activity toward RNA and dinucleotides by no more than 50% and produce slight increases in preference for adenosine vs guanosine at position N' of NpN' substrates. Mutations of Ser-118 to Asp and Arg have a larger effect, decreasing activity by factors of approximately 2 and 4, respectively, toward all substrates examined. These results indicate that: (i) Thr-44 is important for recognition of the pyrimidine moiety at position N, (ii) Glu-108 may make a small contribution to binding the N'-nucleotide, and (iii) Ser-118 has a minor functional role, which appears to involve catalysis rather than nucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Curran
T P
TP
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Shapiro
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R
Riordan
J F
JF
D043925:Angiogenesis Inducing Agents / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
D043925:Angiogenesis Inducing Agents; D005971:Glutamates; D009841:Oligonucleotides; D011506:Proteins; D013912:Threonine; D018698:Glutamic Acid; D012694:Serine; C046366:angiogenin; D012259:Ribonuclease, Pancreatic