Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function

Protein Science : a Publication of the Protein Society
P T BeerninkH K Schachman

Abstract

A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N:-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric. As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper f...Continue Reading

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Citations

Aug 30, 2005·Biomolecular Engineering·Gloria Saab-RincónXavier Soberón
Oct 31, 2002·Protein Expression and Purification·Bernard N ViolandDavid L Zeng
Jan 13, 2004·Biomolecular Engineering·Ronald T Piervincenzi, Ashutosh Chilkoti
May 10, 2001·Protein Science : a Publication of the Protein Society·X Ni, H K Schachman
Apr 13, 2012·PLoS Computational Biology·Spencer Bliven, Andreas Prlić
Feb 4, 2005·Proceedings of the National Academy of Sciences of the United States of America·Ramon K TabtiangRobert T Sauer
Jan 13, 2015·Analytical Biochemistry·Brennal PierreJin Ryoun Kim
Sep 19, 2007·Chembiochem : a European Journal of Chemical Biology·Zhen QianStefan Lutz
Jun 11, 2015·Protein Science : a Publication of the Protein Society·Jason H WhitfieldColin J Jackson
Sep 30, 2005·Journal of the American Chemical Society·Zhen Qian, Stefan Lutz
Dec 28, 2010·Archives of Biochemistry and Biophysics·Grzegorz PiszczekAlan Peterkofsky

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