Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability

Biochimica Et Biophysica Acta
Teisuke TakitaKuniyo Inouye

Abstract

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabiliz...Continue Reading

References

Jul 26, 1968·Biochemical and Biophysical Research Communications·J Feder
Aug 1, 1970·European Journal of Biochemistry·K Morihara, H Tsuzuki
Nov 23, 1971·Biochemistry·J FederB S Wildi
Oct 8, 1969·Biochemical and Biophysical Research Communications·S A LattB L Vallee
Oct 5, 1982·Journal of Molecular Biology·M A Holmes, B W Matthews
Apr 25, 1997·The Journal of Biological Chemistry·J MansfeldV G Eijsink
Feb 23, 2002·The Journal of Biological Chemistry·Arno de KreijJens E Nielsen
May 31, 2002·Acta Crystallographica. Section D, Biological Crystallography·Andrew C Hausrath, Brian W Matthews
Aug 24, 2004·Bioscience, Biotechnology, and Biochemistry·Hiroshi OnedaKuniyo Inouye
Sep 24, 2004·Journal of Biotechnology·Vincent G H EijsinkGert Vriend
Jun 3, 2005·Acta Crystallographica. Section D, Biological Crystallography·Masayuki KamoMasaru Tanokura
Oct 12, 2005·Protein Expression and Purification·Kiyoshi YasukawaKuniyo Inouye
Apr 5, 2007·Journal of Biochemistry·Yasuhiko Hashida, Kuniyo Inouye
Sep 18, 2007·Biochimica Et Biophysica Acta·Kiyoshi Yasukawa, Kuniyo Inouye
Jul 12, 1972·Nature: New Biology·K TitaniH Neurath

❮ Previous
Next ❯

Citations

Oct 23, 2012·Journal of Biochemistry·Yuichi KawasakiKuniyo Inouye
May 1, 2010·Toxins·Frank J LebedaMichael Adler
Dec 8, 2009·Neuroscience Letters·Catarina Moreira PinhoCaroline Graff

❮ Previous
Next ❯

Related Concepts

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.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

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.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.