Site-directed mutagenesis of human soluble calcium-activated nucleotidase 1 (hSCAN-1): identification of residues essential for enzyme activity and the Ca(2+)-induced conformational change

Biochemistry
Mingyan Yang, T L Kirley

Abstract

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) is the human homologue of soluble apyrases found in blood-sucking insects. This family of nucleotidases is unrelated in sequence to more well-studied nucleotidases, and very little is known about the enzymatic mechanism. By multiple sequence alignment, eight regions that are highly conserved in the hSCAN-1 family were identified and named. To identify amino acids important for catalytic activity and enzyme specificity, seven point mutations were constructed, expressed in bacteria, refolded, purified, and characterized. Substitution of glutamic acid 130 with tyrosine resulted in dramatically increased nucleotidase activities, while mutagenesis of aspartic acid 151 to alanine and aspartic acid 84 to alanine completely abolished activity. Mutagenesis of arginine 133 and arginine 271 resulted in enzymes with very little nucleotidase activity. Mutagenesis of aspartic acid 175 to alanine and glycine 122 to glutamic acid had smaller negative effects on enzyme activities. Previously, our laboratory showed that calcium triggers a conformational change in hSCAN-1 necessary for nucleotidase activity. Here we show that several mutants (D84A, R133A, and D151A) that lost most of their ...Continue Reading

References

Dec 20, 1996·The Journal of Biological Chemistry·E KaczmarekS C Robson
Mar 15, 1997·The Journal of Clinical Investigation·A J MarcusC R Maliszewski
Aug 10, 2000·Analytical Biochemistry·J C Krupa, J S Mort
Jul 4, 2001·Structure·E I ScharffH Rüterjans
Aug 9, 2002·The Journal of Biological Chemistry·Bernd U FailerHerbert Zimmermann
Mar 22, 2003·The Journal of Pharmacology and Experimental Therapeutics·Aaron J MarcusRoberto Levi
Apr 5, 2003·The Journal of Pharmacology and Experimental Therapeutics·Casilde SestiRoberto Levi

❮ Previous
Next ❯

Citations

Aug 30, 2012·Journal of the American Chemical Society·David W RooklinYingkai Zhang
Jun 10, 2011·European Journal of Human Genetics : EJHG·Franco LacconeHelga Rehder
Mar 31, 2006·Journal of Enzyme Inhibition and Medicinal Chemistry·S Sumathi, Dipak Dasgupta

❮ 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.