Redesign of catalytic center of an enzyme: aspartic to serine proteinase

Biochemical and Biophysical Research Communications
Takuji Tanaka, Rickey Y Yada

Abstract

In an attempt to convert an aspartic proteinase into another class of proteinase, the catalytic residues of porcine pepsin were substituted with the catalytic triad characteristic of a serine proteinase, using trypsin as the model. Computer modeling suggested six possible sites within porcine pepsin sequence for the introduction of the catalytic triad. The six mutants of pepsin were subsequently constructed and examined for their catalytic activities. Among the six mutants, two mutants, D32S/I300H/G302D (MutI) and D32G/S35H/Y75S/I120D (MutJ), showed peptide hydrolysis activities. In comparison to the original activity of pepsin, the kinetic constants of these mutants were very low with K(m) values of 4.10 and 2.10mM, and k(0) values of 22.2 and 18.0 min(-1). In the presence of PMSF, a serine proteinase inhibitor, the activities for these mutants were inhibited by 86.5% and 80.1%, respectively, indicating that the catalytic triad of the trypsin had been successfully introduced into porcine pepsin.

References

Aug 1, 1992·Acta Crystallographica. Section B, Structural Science·L ChenC Abad-Zapatero
Mar 6, 1992·Science·L HedstromW J Rutter
Oct 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·K SugunaD R Davies
Oct 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·M N JamesT Hofmann
Apr 15, 1996·The Biochemical Journal·T Tanaka, R Y Yada
Apr 15, 1997·Archives of Biochemistry and Biophysics·T Tanaka, R Y Yada
Sep 12, 2000·Current Opinion in Structural Biology·F CedroneE Quéméneur
Jan 9, 2001·Trends in Biotechnology·R Chen
Nov 17, 2001·Chemical Reviews·T M Penning, J M Jez
Jun 1, 2002·Current Opinion in Chemical Biology·Daniel N BolonStephen L Mayo
Mar 13, 2003·The Biochemical Journal·Kenneth G PayieRickey Y Yada
Sep 2, 2003·Current Opinion in Structural Biology·Paul A Dalby
Nov 12, 2003·Biochemistry·Brian C BryksaRickey Y Yada

❮ Previous
Next ❯

Citations

Jul 19, 2006·Cellular & Molecular Biology Letters·Ajay SrinivasanVidya S Gupta

❮ 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.