Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

Protein Science : a Publication of the Protein Society
G I YakovlevA A Makarov

Abstract

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and th...Continue Reading

References

Aug 1, 1976·Archives of Biochemistry and Biophysics·M Zabinski, Frederick G Walz
Nov 1, 1978·Proceedings of the National Academy of Sciences of the United States of America·A Warshel
Feb 15, 1992·European Journal of Biochemistry·G I YakovlevJozef Sevcík
Apr 5, 1992·Journal of Molecular Biology·G KoellnerW Saenger
Jun 5, 1992·Journal of Molecular Biology·E M MeieringA R Fersht
Jan 1, 1972·Cold Spring Harbor Symposia on Quantitative Biology·R J Williams
Jan 17, 1995·Proceedings of the National Academy of Sciences of the United States of America·Brian K ShoichetB W Matthews
Jan 7, 1998·Protein Expression and Purification·E J HebertC Nick Pace
Jun 24, 1998·Journal of Molecular Biology·C Nick PaceGerald R Grimsley
Sep 24, 1999·Protein Science : a Publication of the Protein Society·Gerald R GrimsleyC Nick Pace
May 11, 2000·Proceedings of the National Academy of Sciences of the United States of America·J A EndrizziH K Schachman
Aug 10, 2000·Protein Science : a Publication of the Protein Society·C Nick PaceKevin L Shaw
Nov 18, 2000·Structure·T KajanderA Goldman
May 23, 2001·Protein Science : a Publication of the Protein Society·Kevin L ShawC Nick Pace
Jul 17, 2001·Proteins·Douglas V LaurentsMarta Bruix
Sep 14, 2001·Journal of Molecular Biology·C Nick PaceJozef Sevcík
Jul 24, 2002·Acta Crystallographica. Section D, Biological Crystallography·Jozef SevcíkKeith S Wilson
Jul 30, 2002·Journal of Molecular Biology·Beth M Beadle, Brian K Shoichet
Sep 18, 2002·Archives of Biochemistry and Biophysics·Betty Jo Chitester, Frederick G Walz
Nov 6, 2002·Biochemistry·Sharon Hammes-Schiffer
Nov 8, 2002·Journal of Molecular Biology·Gail J BartlettJanet M Thornton
Jan 16, 2003·Journal of Molecular Biology·Beatrice M P Huyghues-DespointesJ Martin Scholtz
Mar 1, 1996·Acta Crystallographica. Section D, Biological Crystallography·J SevcikK S Wilson

Citations

Jan 22, 2004·Biochemical and Biophysical Research Communications·Olga N IlinskayaA A Makarov
Feb 16, 2012·The Journal of Biological Chemistry·Brook E HeatonSean Crosson
Nov 18, 2005·Journal of Molecular Biology·Saul TreviñoC Nick Pace
Jul 25, 2009·Proteins·Hailong FuC Nick Pace
Mar 27, 2010·Protein Science : a Publication of the Protein Society·Hailong FuC Nick Pace

Related Concepts

Ribonuclease Sa2
Arginine hydrochloride
Calorimetry, Differential Scanning
Catalysis
Enzyme Stability
D-Glutamine
Histidine
Hydrogen-Ion Concentration
Alloenzymes
Poly I

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Neural Activity: Imaging

Imaging of neural activity in vivo has developed rapidly recently with the advancement of fluorescence microscopy, including new applications using miniaturized microscopes (miniscopes). This feed follows the progress in this growing field.

The Tendon Seed Network

Tendons are rich in the extracellular matrix and are abundant throughout the body providing essential roles including structure and mobility. The transcriptome of tendons is being compiled to understand the micro-anatomical functioning of tendons. Discover the latest research pertaining to the Tendon Seed Network here.

Myocardial Stunning

Myocardial stunning is a mechanical dysfunction that persists after reperfusion of previously ischemic tissue in the absence of irreversible damage including myocardial necrosis. Here is the latest research.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Incretins

Incretins are metabolic hormones that stimulate a decrease in glucose levels in the blood and they have been implicated in glycemic regulation in the remission phase of type 1 diabetes. Here is the latest research.

Chromatin Regulation and Circadian Clocks

The circadian clock plays an important role in regulating transcriptional dynamics through changes in chromatin folding and remodelling. Discover the latest research on Chromatin Regulation and Circadian Clocks here.

Long COVID-19

“Long Covid-19” describes illness in patients who are reporting long-lasting effects of the SARS-CoV-19 infection, often long after they have recovered from acute Covid-19. Ongoing health issues often reported include low exercise tolerance and breathing difficulties, chronic tiredness, and mental health problems such as post-traumatic stress disorder and depression. This feed follows the latest research into Long Covid.

Spatio-Temporal Regulation of DNA Repair

DNA repair is a complex process regulated by several different classes of enzymes, including ligases, endonucleases, and polymerases. This feed focuses on the spatial and temporal regulation that accompanies DNA damage signaling and repair enzymes and processes.