Identification of enzyme-substrate and enzyme-product complexes in the catalytic mechanism of glucoamylase from Aspergillus awamori

Biochemistry
S Natarajan, M R Sierks

Abstract

Intermediates in the catalytic mechanism of Aspergillus awamori glucoamylase (GA) were identified by studying pre-steady-state and steady-state kinetics of the wild-type GA/maltose and Trp120 -->Phe GA/maltotriose reactions in H2O and D2O. Pre-steady-state fluorescence signal analysis was carried out to ascertain the relative intrinsic fluorescence of the enzyme intermediates. A three-step minimal pathway for oligosaccharide hydrolysis represented by E + Gx (k1) reversible (k-1) EGX (k2)reversible(k-2) EP (kcat)--> E + P is proposed. The first step, represented by the association constant K1 (k1/k-1), depicts the fast formation of enzyme-substrate complex and is the primary factor in fluorescence quenching. A 2.7-fold increase in K1 with D2O as solvent is observed with both enzymes due to the cumulative effect of deuterium on complex hydrogen bonding at the active site. The second step further quenches the enzyme fluorescence and is identified as the hydrolytic step, forming an enzyme-product complex. Both k2 and k-2 values show similar 2-fold decreases in D2O for both enzymes, consistent with the microscopic reversibility of the hydrolytic reaction. The solvent isotopic effect on the hydrolytic step is likely due to either abs...Continue Reading

References

Jan 1, 1986·Annual Review of Biochemistry·F A Quiocho
Jan 1, 1983·Molecular and Cellular Biochemistry·K HiromiA Tanaka
May 13, 1994·Journal of Molecular Biology·A E AleshinR B Honzatko

❮ Previous
Next ❯

Citations

Jan 11, 2001·Biochimica Et Biophysica Acta·J SauerB Svensson
Aug 17, 1999·Current Opinion in Biotechnology·C Ford
Jun 29, 2000·Annual Review of Biochemistry·H D Ly, S G Withers
Dec 14, 1999·Bioscience, Biotechnology, and Biochemistry·A TanakaH Obata
Oct 26, 2005·Journal of Molecular Biology·T CollinsG Feller
Aug 28, 2009·Critical Reviews in Biotechnology·Pardeep Kumar, T Satyanarayana

❮ 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.