Structural features that determine the enzymatic potency and specificity of human angiogenin: threonine-80 and residues 58-70 and 116-123.
Human angiogenin (Ang), a homologue of bovine pancreatic ribonuclease A (RNase A), is a potent inducer of blood vessel formation. It exerts a ribonucleolytic activity that is 10(5)-10(6)-fold lower than that of RNase A but nonetheless essential for biological action. Previous studies revealed some of the structural features of Ang that underlie its catalytic inefficiency: Gln-117 blocks the space corresponding to the pyrimidine binding site of RNase A and Ang lacks the disulfide loop 65-72 that forms most of the purine binding site of RNase A. Additional features have now been identified by mutagenesis and kinetics. Thr-80, which hydrogen-bonds to the pyrimidine-binding residue Thr-44, plays an important part in attenuating activity and in determining pyrimidine specificity: mutation to Ala increases activity toward cytidylyl substrates by 11-15-fold but has only a minimal effect on cleavage of uridylyl substrates. The properties of T44A/T80A and Q117A/T80A double mutants demonstrate that these changes are mediated by Thr-44 and are largely independent of the blockage by Gln-117. The side chain of Ser-118 also suppresses enzymatic activity: S118A is 5-7-fold more effective than Ang. This increase appears to reflect the loss of a hydrogen bond with Asp-116 that helps to orient Gln-117. The effects of deleting residues 119-123 suggest that main-chain atoms of the C-terminal 3(10) helix make a small further contribution. Finally, the significance of the absence of the RNase A loop 65-72 from Ang has been investigated by reexamining the earlier derivative ARH-I (in which Ang residues 58-70 have been replaced by residues 59-73 of RNase) and generating new derivatives of this hybrid protein. The results suggest that the RNase A segment of ARH-I not only provides more effective purine recognition but also counteracts the deleterious effects of Gln-117 and Thr-80 on the pyrimidine site.
Shapiro
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Center for Biochemical and Biophysical Sciences and Medicine, Boston, Massachusetts 02115, USA. shapiro@ferret.med.harvard.edu
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000235:genetics; D013912:Threonine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013912:Threonine; C046366:angiogenin; D012259:Ribonuclease, Pancreatic