A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity? |
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Authors: | Allemann Rudolf K; Presnell Scott R; Benner Steven A |
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Affiliation: | Laboratory for Organic Chemistry, Swiss Federal Institute of Technology CH-8092 Zurich, Switzerland |
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Abstract: | A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 6374 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 5970 of RNase were inserted into angiogeninHarper and Vallee (1989) Biochemistry, 28, 18751884].Thus, the external loop in residues 6374 of RNase A appearsto behave, at least in part, as an interchangeable modulethat influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein. |
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Keywords: | angiogenin/ evolutionary guidance/ folding pathway/ ribonuclease/ site-directed mutagenesis |
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