A search for single substitutions that eliminate enzymatic function in a bacterial ribonuclease - PubMed (original) (raw)
. 1998 May 19;37(20):7157-66.
doi: 10.1021/bi9804028.
Affiliations
- PMID: 9585527
- DOI: 10.1021/bi9804028
A search for single substitutions that eliminate enzymatic function in a bacterial ribonuclease
D D Axe et al. Biochemistry. 1998.
Abstract
Exhaustive-substitution studies, where many amino acid replacements are individually tested at all positions in a natural protein, have proven to be very valuable in probing the relationship between sequence and function. The broad picture that has emerged from studies of this sort is one of functional tolerance of substitution. We have applied this approach to barnase, a 110-residue bacterial ribonuclease. Because the selection system used to score barnase mutants as active or inactive detects activity down to a level that can be approached by nonenzyme catalysts, mutants that test inactive are essentially devoid of enzymatic function. Of the 109 barnase positions subjected to substitution, only 15 (14%) are vulnerable to this extreme level of inactivation, and only 2 could not be substituted without such inactivation. A total of 33 substitutions (amounting to 5% of the explored substitutions) were found to render barnase wholly inactive. The profoundly disruptive effects of all of these inactivating substitutions appear to result from either (1) replacement of a side chain that is directly involved in substrate binding or catalysis, (2) replacement of a substantially buried side chain, (3) introduction of a proline residue, or (4) replacement of a glycine residue. Although substitutions of these types are functionally tolerated more often than not, the system used here indicates that only these sorts of substitution are capable of single-handedly reducing catalytic function to, or nearly to, levels that can be achieved by nonenzyme catalysts.
Similar articles
- An irregular beta-bulge common to a group of bacterial RNases is an important determinant of stability and function in barnase.
Axe DD, Foster NW, Fersht AR. Axe DD, et al. J Mol Biol. 1999 Mar 12;286(5):1471-85. doi: 10.1006/jmbi.1999.2569. J Mol Biol. 1999. PMID: 10064710 - X-ray structural analysis of compensating mutations at the barnase-barstar interface.
Martin C, Hartley R, Mauguen Y. Martin C, et al. FEBS Lett. 1999 Jun 11;452(3):128-32. doi: 10.1016/s0014-5793(99)00621-3. FEBS Lett. 1999. PMID: 10386576 - Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.
Jucovic M, Hartley RW. Jucovic M, et al. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2343-7. doi: 10.1073/pnas.93.6.2343. Proc Natl Acad Sci U S A. 1996. PMID: 8637875 Free PMC article. - Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation.
Frisch C, Schreiber G, Johnson CM, Fersht AR. Frisch C, et al. J Mol Biol. 1997 Apr 4;267(3):696-706. doi: 10.1006/jmbi.1997.0892. J Mol Biol. 1997. PMID: 9126847 - In vivo system for the detection of low level activity barnase mutants.
Jucovic M, Hartley RW. Jucovic M, et al. Protein Eng. 1995 May;8(5):497-9. doi: 10.1093/protein/8.5.497. Protein Eng. 1995. PMID: 8532672
Cited by
- Enriching productive mutational paths accelerates enzyme evolution.
Patsch D, Schwander T, Voss M, Schaub D, Hüppi S, Eichenberger M, Stockinger P, Schelbert L, Giger S, Peccati F, Jiménez-Osés G, Mutný M, Krause A, Bornscheuer UT, Hilvert D, Buller RM. Patsch D, et al. Nat Chem Biol. 2024 Sep 11. doi: 10.1038/s41589-024-01712-3. Online ahead of print. Nat Chem Biol. 2024. PMID: 39261644 - Computational scoring and experimental evaluation of enzymes generated by neural networks.
Johnson SR, Fu X, Viknander S, Goldin C, Monaco S, Zelezniak A, Yang KK. Johnson SR, et al. Nat Biotechnol. 2024 Apr 23. doi: 10.1038/s41587-024-02214-2. Online ahead of print. Nat Biotechnol. 2024. PMID: 38653796 - Inverse folding of protein complexes with a structure-informed language model enables unsupervised antibody evolution.
Shanker VR, Bruun TUJ, Hie BL, Kim PS. Shanker VR, et al. bioRxiv [Preprint]. 2023 Dec 21:2023.12.19.572475. doi: 10.1101/2023.12.19.572475. bioRxiv. 2023. PMID: 38187780 Free PMC article. Updated. Preprint. - LibGENiE - A bioinformatic pipeline for the design of information-enriched enzyme libraries.
Patsch D, Eichenberger M, Voss M, Bornscheuer UT, Buller RM. Patsch D, et al. Comput Struct Biotechnol J. 2023 Sep 14;21:4488-4496. doi: 10.1016/j.csbj.2023.09.013. eCollection 2023. Comput Struct Biotechnol J. 2023. PMID: 37736300 Free PMC article. - Extreme genetic fragility of the HIV-1 capsid.
Rihn SJ, Wilson SJ, Loman NJ, Alim M, Bakker SE, Bhella D, Gifford RJ, Rixon FJ, Bieniasz PD. Rihn SJ, et al. PLoS Pathog. 2013;9(6):e1003461. doi: 10.1371/journal.ppat.1003461. Epub 2013 Jun 20. PLoS Pathog. 2013. PMID: 23818857 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources