Pyridoxamine-pyruvate transaminase. I. Determination of the active site stoichiometry and the pH dependence of the dissociation constant for 5'-deoxypyridoxal

Biochemistry ◽  
1977 ◽  
Vol 16 (24) ◽  
pp. 5241-5246 ◽  
Author(s):  
Penny J. Gilmer ◽  
William S. McIntire ◽  
Jack F. Kirsch
Keyword(s):  
Dissociation Constant ◽  
Active Site ◽  
Ph Dependence

scholarly journals Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes

1983 ◽  
Vol 215 (2) ◽  
pp. 287-294 ◽  
Author(s):  
R R Cook ◽  
J C Powers
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Activated Protein C ◽  
Factor Xa ◽  
Bovine Thrombin ◽  
Rapid Formation ◽  
Factor Xiia ◽  
Chymotrypsin A ◽  
Ellman's Reagent

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4′-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4′-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.

scholarly journals Probing the ionization state of substrate α-d-glucopyranosyl phosphate bound to glycogen phosphorylase b

1995 ◽  
Vol 308 (3) ◽  
pp. 1017-1023 ◽  
Author(s):  
I P Street ◽  
S G Withers
Keyword(s):  
Active Site ◽  
Glycogen Phosphorylase ◽  
Ph Dependence ◽  
Substrate Inhibition ◽  
19F Nmr ◽  
Ionization State ◽  
Nmr Studies ◽  
Substrate Analogues ◽  
Glycogen Phosphorylase B ◽  
Pka Value

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

scholarly journals Synthesis of Fluorescent Carbon Dots and Their Application in Ascorbic Acid Detection

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1246
Author(s):  
Tengfei Wang ◽  
Hui Luo ◽  
Xu Jing ◽  
Jiali Yang ◽  
Meijun Huo ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Fluorescence Intensity ◽  
Carbon Dots ◽  
Ph Dependence ◽  
Scale Up ◽  
Water Soluble ◽  
Jujube Fruit ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Water-soluble fluorescent carbon dots (CDs) were synthesized by a hydrothermal method using citric acid as the carbon source and ethylenediamine as the nitrogen source. The repeated and scale-up synthetic experiments were carried out to explore the feasibility of macroscopic preparation of CDs. The CDs/Fe3+ composite was prepared by the interaction of the CDs solution and Fe3+ solution. The optical properties, pH dependence and stability behavior of CDs or the CDs/Fe3+ composite were studied by ultraviolet spectroscopy and fluorescence spectroscopy. Following the principles of fluorescence quenching after the addition of Fe3+ and then the fluorescence recovery after the addition of asorbic acid, the fluorescence intensity of the carbon dots was measured at λex = 360 nm, λem = 460 nm. The content of ascorbic acid was calculated by quantitative analysis of the changing fluorescence intensity. The CDs/Fe3+ composite was applied to the determination of different active molecules, and it was found that the composite had specific recognition of ascorbic acid and showed an excellent linear relationship in 5.0–350.0 μmol·L−1. Moreover, the detection limit was 3.11 μmol·L−1. Satisfactory results were achieved when the method was applied to the ascorbic acid determination in jujube fruit. The fluorescent carbon dots composites prepared in this study may have broad application prospects in a rapid, sensitive and trace determination of ascorbic acid content during food processing.

Statistical Description of Isotope Exchange Processes: A Multisite Model for the 18O Exchange

1982 ◽  
Vol 37 (11-12) ◽  
pp. 1161-1169 ◽  
Author(s):  
Paul Rösch
Keyword(s):  
Active Site ◽  
Isotope Exchange ◽  
Analytical Procedure ◽  
Matrix Formalism ◽  
Model Calculations ◽  
Exchange Processes ◽  
Reaction Solution ◽  
Different Types ◽  
Refinement Procedure

Abstract An analytical procedure has been developed for the determination of isotope exchange processes as exemplified by the 18O exchange catalysed by enzyme-nucleotide complexes. The model is able to handle more than one type of active site per reaction solution and is also able to distinguish between different types of inequivalence of the oxygens of enzyme bound Pi. Use of transition matrix formalism and basic statistical considerations lead directly to the simple model. A data refinement procedure is introduced and model calculations are shown.

scholarly journals pH-controlled hydrogen-bonding (Short Communication)

1974 ◽  
Vol 143 (3) ◽  
pp. 775-777 ◽  
Author(s):  
John L. Wood
Keyword(s):  
Hydrogen Bonding ◽  
Dissociation Constant ◽  
Small Proportion ◽  
Association Constant ◽  
Short Communication ◽  
Dibasic Acid ◽  
Ph Dependence ◽  
Ph Values ◽  
Conjugate Acid ◽  
Total Base

The pH-dependence of the degree of hydrogen-bonding between a base and its conjugate acid is considered. When only a small proportion of the total base is complexed, the amount complexed is proportional to (1+coshp)−1 where p=2.303 (pKa–pH), pKa being the dissociation constant of the conjugate acid. This represents sharp pH-dependence. As the proportion complexed increases, the curve broadens, eventually becoming flat-topped, with more than half the base complexed over the range of pH values pKa±logKC, approximately. (K is the complex association constant and C is the formal base concentration, including all forms.) There are similarities to the extent of mono-protonation of a dibasic acid.

Nonlinear Vibrations in a 2-Dimensional Protein Cluster Model with Linear Bonds

2000 ◽  
Vol 214 (1) ◽  
Author(s):  
E.G. Shidlovskaya ◽  
L. Schimansky-Geier ◽  
Yu.M. Romanovsky
Keyword(s):  
Active Site ◽  
Internal Resonance ◽  
Cluster Model ◽  
Active Sites ◽  
Nonlinear Vibrations ◽  
Nonlinear Phenomena ◽  
Two Dimensional ◽  
Dimensional Model ◽  
Two Dimensional Model

A two dimensional model for the substrate inside a pocket of an active site of an enzyme is presented and investigated as a vibrational system. The parameters of the system are evaluated for α-chymotrypsin. In the case of internal resonance it is analytically and numerically shown that the energy concentrated on a certain degree of freedom might be several times larger than in the non-resonant case. Additionally, the system is driven by harmonic excitations and again energy due to nonlinear phenomena is redistributed inhomogeneously. These results may be of importance for the determination of the rates of catalytic events of substrates bound in pockets of active sites.

Potentiometric determination of ionizations at the active site of papain

Biochemistry ◽  
1976 ◽  
Vol 15 (23) ◽  
pp. 5009-5017 ◽  
Author(s):  
Sidney D. Lewis ◽  
Frederick A. Johnson ◽  
Jules A. Shafer
Keyword(s):  
Active Site ◽  
Potentiometric Determination
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