scholarly journals The modification of cholinesterase activity by 5, 5′-dithiobis-(2-nitrobenzoic acid) included in the coupled spectrophotometric assay. Evidence for a non-catalytic substrate-binding site

1973 ◽  
Vol 131 (2) ◽  
pp. 369-374 ◽  
Author(s):  
C. Brownson ◽  
D. C. Watts
Keyword(s):  
Binding Site ◽  
Cholinesterase Activity ◽  
Substrate Binding ◽  
Spectrophotometric Assay ◽  
Substrate Binding Site ◽  
Nitrobenzoic Acid ◽  
Quantitative Measurements ◽  
Km Value ◽  
Hill Coefficients ◽  
A Site

1. Compared with the acetylcholinesterase assay carried out in the absence of a dithiol, the presence of 5,5′-dithiobis-(2-nitrobenzoic acid) caused marked activation, 6,6′-dithiodinicotinic acid and 2,2′-dithiobis-(5-nitropyridine) less so and 2,2′-dithiodipyridine (aldrithiol-2) had no effect at all. Measurements are further complicated in that the 5-thio-2-nitrobenzoate ion also appears to interact with the enzyme, resulting in slightly lowered absorbance values. 2. Acetylthiocholine competes for the 5,5′-dithiobis-(2-nitrobenzoic acid)-binding site so that activation is essentially eliminated by saturating concentrations of substrate. The presence of the dithiol decreases the Km value of acetylthiocholine. 3. Similar results were obtained with pseudocholinesterase. However, with butyrylthiocholine clear activation was still observed under Vmax. conditions in addition to Km being lowered. 4. All the data yielded Hill coefficients of 1 and analysis of the results leads to the conclusion that activation results from the dithiol being bound to a site on the subunit that is actively catalysing ester hydrolysis. 5. The use of aldrithiol-2 is recommended for kinetic work where absolute quantitative measurements are required.

Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution

2010 ◽  
Vol 431 (3) ◽  
pp. 401-412 ◽  
Author(s):  
Yumewo Suzuki ◽  
Kuniko Asada ◽  
Junichi Miyazaki ◽  
Takeo Tomita ◽  
Tomohisa Kuzuyama ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Binding Site ◽  
Directed Evolution ◽  
Substrate Binding ◽  
Dna Shuffling ◽  
Turnover Number ◽  
Substrate Binding Site ◽  
Km Value ◽  
Isopropylmalate Dehydrogenase

HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB−) to grow on a minimal medium in 2 days were obtained. One of the variants LR5–1, with eight amino acid replacements, was found to possess a 65-fold increased kcat/Km value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the kcat/Km value and a partial recovery of the decreased thermotolerance of LR5–1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5–1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5–1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.

scholarly journals Kinetic studies on pantothenase from Pseudomonas fluorescens. Effects of pH on substrate and inhibitor binding

1976 ◽  
Vol 157 (2) ◽  
pp. 415-421 ◽  
Author(s):  
K Airas
Keyword(s):  
Binding Site ◽  
Phosphate Buffer ◽  
Substrate Binding ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Inhibitor Binding ◽  
Substrate Binding Site ◽  
Ph Values ◽  
Km Value ◽  
Rate Profile

The velocity of the pantothenase-catalysed hydrolysis of pantothenate was studied over pH5.5-9, and in the presence of oxalate or oxaloacetate as an inhibitor. The pH-dependence of the reaction can be described by a kinetic equation containing two ionizations of the enzyme, with one ionizable group located at the substrate-binding site, and the other at the inhibitor-binding site. The Km value of pantothenase to pantothenate depends on the buffer used, and phosphate tends to give somewhat lower values than other buffers. Km also depends on pH, the best activities being observed at basic pH values. The pH-independent Km is 7.6mM in phosphate buffer at 20 degrees C; the corresponding Kapp.m value at pH7 is 15 mM. The pK value of the ionizable group at the substrate-binding site was measured by two methods: from the pH-rate profile and from the pH-Km rofile. pK is 7.0 in phosphate buffer at 20 degrees C, ranging in various buffers between 6.9 and 7.3. The van't Hoff enthalpies of substrate binding and H+ ion binding were—14kJ/mol respectively. The inhibition by oxalate or oxaloacetate is of non-competitive type and depends on pH, the inhibitors being effective at acidic pH values. The pK value of the ionizable group at the inhibitor-binding site was derived from the measurements of the K1 values over the pH range 6-7.5. The pK value was 6.4 in oxaloacetate inhibition, the pH-independent K1 being 0.36mM, and the corresponding Kapp.m about 1.8mM at pH7. Phenylmethanesulphonyl fluoride was capable of inactivating pantothenase.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

2020 ◽  
Vol 21 (2) ◽  
pp. 117-130 ◽  
Author(s):  
Mohammad J. Hosen ◽  
Mahmudul Hasan ◽  
Sourav Chakraborty ◽  
Ruhshan A. Abir ◽  
Abdullah Zubaer ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Glucose Transporter ◽  
Substrate Binding ◽  
Mutation Screening ◽  
Homology Model ◽  
Connective Tissue Disorder ◽  
Crystallographic Structure ◽  
Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

The role of Glu196 in the environment around the substrate binding site of leucine aminopeptidase fromStreptomyces griseus

FEBS Letters ◽  
2006 ◽  
Vol 580 (3) ◽  
pp. 912-917 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Misugi Uraji ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Binding Site ◽  
Leucine Aminopeptidase ◽  
Substrate Binding ◽  
Substrate Binding Site
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