scholarly journals Kinetic and chemical mechanisms for the effects of univalent cations on the spectral properties of aromatic amine dehydrogenase

1998 ◽  
Vol 329 (1) ◽  
pp. 175-182 ◽  
Author(s):  
Zhenyu ZHU ◽  
L. Victor DAVIDSON
Keyword(s):  
Absorption Spectrum ◽  
Reaction Mechanism ◽  
Aromatic Amine ◽  
Ph Dependence ◽  
Spectral Change ◽  
Univalent Cations ◽  
Prosthetic Group ◽  
Cation Concentration ◽  
Amine Dehydrogenase ◽  
Spectral Perturbation

Univalent cations and pH influence the UV-visible absorption spectrum of the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH). Little spectral perturbation was observed when pH was varied in the absence of univalent cations. The addition of alkali metal univalent cations (K+, Na+, Li+, Rb+ or Cs+) to oxidized AADH caused significant changes in its absorption spectrum. The apparent Kd for each cation, determined from titrations of the spectral perturbation, decreased with increasing pH. Transient kinetic studies involving rapid mixing of AADH with cations and pH jump revealed that the rate of the cation-induced spectral changes initially decreased with increasing cation concentration to a minimum value, then increased with increasing cation concentration. A kinetic model was developed to fit these data, determine the true pH-independent Kd values for K+ and Na+, and explain the pH-dependence of the apparent Kd. A chemical reaction mechanism, based on the kinetic data, is presented in which the metallic univalent cation facilitates the chemical modification of the TTQ prosthetic group to form an hydroxide adduct which gives rise to the spectral change. Addition of NH4+/NH3 to AADH caused changes in the absorption spectrum which were very different form those caused by addition of the metallic univalent cations. The kinetics of the reaction induced by addition of NH4+/NH3 were also different, being simple saturation kinetics. Another reaction mechanism is proposed for the NH4+/NH3-induced spectral change that involves nucleophilic addition of the unprotonated NH3 to TTQ. The general relevance of these data and models to the physiological reactions of TTQ-dependent enzymes and to the roles of univalent cations in modulating enzyme activity are discussed.

scholarly journals New Insights into the Reductive Half-reaction Mechanism of Aromatic Amine Dehydrogenase Revealed by Reaction with Carbinolamine Substrates

2007 ◽  
Vol 282 (33) ◽  
pp. 23766-23777 ◽  
Author(s):  
Anna Roujeinikova ◽  
Parvinder Hothi ◽  
Laura Masgrau ◽  
Michael J. Sutcliffe ◽  
Nigel S. Scrutton ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Aromatic Amine ◽  
Amine Dehydrogenase

Temperature Gradients Through EHD Films and Molecular Alignment Evidenced By Infrared Spectroscopy

1981 ◽  
Vol 103 (1) ◽  
pp. 65-73 ◽  
Author(s):  
V. W. King ◽  
J. L. Lauer
Keyword(s):  
Absorption Spectrum ◽  
Emission Spectra ◽  
Infrared Emission ◽  
Molecular Alignment ◽  
Temperature Gradients ◽  
Spectral Change ◽  
Continuum Source ◽  
Polyphenyl Ether ◽  
Elementary Analysis ◽  
Gap Width

Partial and complete emission band reversals have been observed in the infrared emission spectra from portions of operating sliding contacts. An elementary analysis has been carried out to show that partial reversals are due to temperature gradients in the fluid film—the film acts both as a radiation-emitter and absorber, and that total reversals—an emission spectrum appears as an absorption spectrum—are likely to be due to a continuum source, such as hot solid asperities. The total energy radiated under the latter conditions exceeds that under the others. A decrease in gap width with increased load was accompanied by a dramatic spectral change in the case of 5P4E polyphenyl ether, which is indicative of molecular alignment.

scholarly journals Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM 1

1971 ◽  
Vol 123 (5) ◽  
pp. 757-771 ◽  
Author(s):  
R. R. Eady ◽  
P. J. Large
Keyword(s):  
Fluorescence Emission ◽  
Electrophoretic Analysis ◽  
Enzyme Mechanism ◽  
Heat Denaturation ◽  
Kinetic Properties ◽  
Microbial Oxidation ◽  
Prosthetic Group ◽  
Iron And Zinc ◽  
Amine Dehydrogenase ◽  
Phenazine Methosulphate

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver–Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.

scholarly journals Physical-chemical properties of C-phycocyanin isolated from an acido-thermophilic eukaryote, Cyanidium caldarium

1975 ◽  
Vol 147 (1) ◽  
pp. 63-70 ◽  
Author(s):  
O H Kao ◽  
M R Edwards ◽  
D S Berns
Keyword(s):  
Absorption Spectrum ◽  
High Temperature ◽  
Amino Acid ◽  
Ph Dependence ◽  
Chemical Properties ◽  
Sedimentation Velocity ◽  
Subunit Structure ◽  
Cyanidium Caldarium ◽  
Eukaryotic Alga ◽  
Physical Chemical

C-Phycocyanin from an acido-thermophilic eukaryotic alga, Cyanidium caldarium, was characterized with respect to subunit structure, absorption spectrum and fluorescence properties and was found to be similar to C-phycocyanins from mesophilic sources. The pH-dependence of fluorescence polarization and the changes in sedimentation velocity as a function of pH, concentration and temperature indicate the presence of extremely large amounts of unusually stable 19S aggregates. It was not possible to disaggregate this phycocyanin completely to monomer under normal conditions. The amino acid composition is similar to that of phycocyanins from other thermophilic and halophilic sources. The isoelectric point of this C-phycocyanin was 5.11, an unusually high value. The properties of this C-phycocyanin suggest an increase in protein stability as its mode of adaptation to the environmental stress of high temperature.

scholarly journals The enzyme aromatic amine dehydrogenase induces a substrate conformation crucial for promoting vibration that significantly reduces the effective potential energy barrier to proton transfer

2008 ◽  
Vol 5 (suppl_3) ◽  
pp. 225-232 ◽  
Author(s):  
Linus O Johannissen ◽  
Nigel S Scrutton ◽  
Michael J Sutcliffe
Keyword(s):  
Potential Energy ◽  
Proton Transfer ◽  
Aromatic Amine ◽  
Effective Potential ◽  
Energy Barrier ◽  
Transfer Event ◽  
Effective Potential Energy ◽  
Potential Energy Barrier ◽  
Mechanical Methods ◽  
Amine Dehydrogenase

The role of promoting vibrations in enzymic reactions involving hydrogen tunnelling is contentious. While models incorporating such promoting vibrations have successfully reproduced and explained experimental observations, it has also been argued that such vibrations are not part of the catalytic effect. In this study, we have employed combined quantum mechanical/molecular mechanical methods with molecular dynamics and potential energy surface calculations to investigate how enzyme and substrate motion affects the energy barrier to proton transfer for the rate-limiting H-transfer step in aromatic amine dehydrogenase (AADH) with tryptamine as substrate. In particular, the conformation of the iminoquinone adduct induced by AADH was found to be essential for a promoting vibration identified previously—this lowers significantly the ‘effective’ potential energy barrier, that is the barrier which remains to be surmounted following collective, thermally equilibrated motion attaining a quantum degenerate state of reactants and products. When the substrate adopts a conformation similar to that in the free iminoquinone, this barrier was found to increase markedly. This is consistent with AADH facilitating the H-transfer event by holding the substrate in a conformation that induces a promoting vibration.

Pressure, Temperature and pH Dependence of the Absorption Spectrum of Reduced Nicotinamide Adenine Dinucleotide

1981 ◽  
Vol 36 (1-2) ◽  
pp. 84-86
Author(s):  
Rainer Jaenicke ◽  
Hans-Dietrich Liidemann ◽  
Gerhard Schmid
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Ph Dependence ◽  
Adiabatic Compression ◽  
Buffer System ◽  
Spectral Change ◽  
Spectral Changes ◽  
High Pressure Studies ◽  
Optimum Wavelength ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
The Given

Abstract Enzymological studies at high hydrostatic pressure generally involve temperature, pH and pressure as variables, owing to the effect of adiabatic compression and the ionization volume of the buffer system. In the case of NAD dependent oxidoreductases this implies that the extinction coefficient of the coenzyme may be affected by p, T and pH, apart from the spectral change accompanying the redox reaction. Measurements of the pressure dependence of the absorbance of NADH show a slight red shift and a 1% decrease (3% increase) of the absorbance at 339 nm (360 nm) at 2 kbar. The pH depen­dence at the given wavelengths amounts to -(2.4 ± 0.1)% per pH unit (25 °C), while the intrinsic temperature effect (after correction for thermal expansion) is of the order of -0.2% per degree (20-30 °C). Applying buffers with negligible ionization volume, 366 nm is the optimum wavelength for high pressure studies up to 2 kbar because here the pressure dependent spectral changes of the NADH absorption vanish.

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