Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups

Protein Science : a Publication of the Protein Society
A R Johnson, E E Dekker

Abstract

L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does n...Continue Reading

References

Apr 1, 1979·Analytical Biochemistry·P W RiddlesB Zerner
Mar 25, 1976·Journal of Molecular Biology·H EklundA Akeson
Aug 1, 1978·Journal of Bacteriology·S KomatsubaraI Chibata
Dec 1, 1992·European Journal of Biochemistry·S Gite, V Shankar
Dec 15, 1991·Analytical Chemistry·F HillenkampB T Chait
Jan 1, 1989·Annals of the New York Academy of Sciences·J R GuestG C Russell
Jul 14, 1989·Biochemical and Biophysical Research Communications·G WolfF Palmieri
Nov 23, 1988·Biochimica Et Biophysica Acta·P A Craig, E E Dekker
Oct 1, 1986·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·U Sinha, J M Brewer
Jan 1, 1972·Methods in Enzymology·K WeberM Osborn
Jul 1, 1983·Archives of Biochemistry and Biophysics·E Ceccarelli, R H Vallejos
Dec 7, 1984·Journal of Chromatography·B A BidlingmeyerT L Tarvin
Jun 1, 1984·Archives of Biochemistry and Biophysics·E M Valle, R H Vallejos
Apr 28, 1995·The Journal of Biological Chemistry·A A KomissarovV G Debabov
Aug 1, 1993·Journal of Protein Chemistry·G B Maralihalli, A S Bhagwat
May 1, 1959·Archives of Biochemistry and Biophysics·G L ELLMAN
Jan 1, 1972·Methods in Enzymology·A F Habeeb

❮ Previous
Next ❯

Citations

Jul 1, 2015·Journal of the American Society for Mass Spectrometry·Zhou PengScott A McLuckey
Jan 1, 1997·Critical Reviews in Biochemistry and Molecular Biology·R N Puri, R W Colman
Aug 12, 2003·Biotechnology and Bioengineering·Hans A KostersHarmen H J de Jongh

❮ 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.

Hereditary Sensory Autonomic Neuropathy

Hereditary Sensory Autonomic Neuropathies are a group of inherited neurodegenerative disorders characterized clinically by loss of sensation and autonomic dysfunction. Here is the latest research on these neuropathies.

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.

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.

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.

Landau-Kleffner Syndrome

Landau Kleffner syndrome (LKS), also called infantile acquired aphasia, acquired epileptic aphasia, or aphasia with convulsive disorder, is a rare childhood neurological syndrome characterized by the sudden or gradual development of aphasia (the inability to understand or express language) and an abnormal electroencephalogram. Discover the latest research on LKS 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.

Regulation of Vocal-Motor Plasticity

Dopaminergic projections to the basal ganglia and nucleus accumbens shape the learning and plasticity of motivated behaviors across species including the regulation of vocal-motor plasticity and performance in songbirds. Discover the latest research on the regulation of vocal-motor plasticity here.