scholarly journals Factors affecting the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the pro-2R and pro-2S protons of glycine

1995 ◽  
Vol 311 (3) ◽  
pp. 1015-1019 ◽  
Author(s):  
J J Milne ◽  
J P G Malthouse
Keyword(s):  
Exchange Reaction ◽  
Catalytic Efficiency ◽  
Second Order ◽  
Beta Subunits ◽  
Enzyme Complex ◽  
Tryptophan Synthase ◽  
Exchange Reactions ◽  
Factors Affecting ◽  
Alpha Subunits ◽  
Beta 2

13C-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20)-catalysed hydrogen-deuterium exchange of the pro-2R and pro-2S protons of [2-13C]glycine. The first- and second-order rate constants for exchange when the alpha 2 beta 2 enzyme complex is or is not saturated with glycine have been determined at pH 7.0 and 7.8. At pH 7.8 the effects of binding the allosteric effector, DL-alpha-glycerol 3-phosphate, and of removing the alpha-subunits have been examined. The beta-subunits preferentially catalyse the exchange of the pro-2R proton of glycine, but adding alpha-subunits decreases the stereospecificity of the exchange reactions. Likewise, binding of DL-alpha-glycerol 3-phosphate to the alpha 2 beta 2 enzyme complex causes a further decrease in the stereospecificity of this reaction. The stereospecificity of the second-order exchange reaction catalysed by the beta-subunits is 136-fold larger than that of the alpha 2 beta 2 enzyme complex in the presence of DL-alpha-glycerol 3-phosphate, while there is only a 5-fold decrease in the stereospecificity of the first-order exchange reaction under the same conditions. We discuss how these results relate to current theories which attempt to explain how the alpha-subunits and DL-alpha-glycerol 3-phosphate modify the catalytic properties of tryptophan synthase.

scholarly journals The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids

1996 ◽  
Vol 314 (3) ◽  
pp. 787-791 ◽  
Author(s):  
John J. MILNE ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
Exchange Rate ◽  
Amino Acid ◽  
Exchange Rates ◽  
Catalytic Efficiency ◽  
Second Order ◽  
Tryptophan Synthase ◽  
Exchange Reactions ◽  
First Order ◽  
Allosteric Effector ◽  
Hydrogen Deuterium

1H-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) catalysed hydrogen–deuterium exchange of the α-protons of L- and D-alanine and -tryptophan. The first-order and second-order rate constants for exchange have been determined at pH 7.8 in the presence and absence of the allosteric effector, DL-α-glycerol 3-phosphate. In the presence of DL-α-glycerol 3-phosphate the stereospecificity of the tryptophan synthase-catalysed first-order exchange rates was in the order tryptophan > alanine > glycine. This increase in stereospecificity was largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged α-proton. A similar increase in the stereospecificity of the second-order exchange rates for alanine was also largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged α-proton of D-alanine. Adding DL-α-glycerol 3-phosphate produced an increase in the stereospecificity of the second-order exchange rate observed with alanine but no significant change in the stereospecificity of the first-order exchange rate with tryptophan. The α-subunits are shown to increase the exchange rates of the α-protons of L-alanine and L-tryptophan. We conclude that the contribution of the R-group of an amino acid to the stereospecificity of the exchange reactions of its α-proton can be similar to or larger than that of its α-carboxylate group. Possible mechanisms that could explain the stereospecificity of these exchange reactions are discussed.

scholarly journals A proton-magnetic-resonance study of hydrogen-exchange reactions of yeast tryptophan synthase

1991 ◽  
Vol 273 (3) ◽  
pp. 605-610
Author(s):  
C J Bailey ◽  
J P G Malthouse
Keyword(s):  
Exchange Reaction ◽  
Hydrogen Exchange ◽  
Ph Dependence ◽  
Proton Exchange ◽  
Free Enzyme ◽  
Tryptophan Synthase ◽  
Exchange Reactions ◽  
Exponential Decay Rate ◽  
Base Form ◽  
Catalytic Constant

1H n.m.r. was used to observe tryptophan formation from indole and L-serine, proton exchange at C-2 of L-tryptophan, and proton exchange at C-2 of L-serine, catalysed by yeast tryptophan synthase in the presence of 2H2O. Tryptophan synthesis took place with compulsory replacement of C-2 hydrogen by solvent hydrogen. The exponential decay rate (kobs) of the serine exchange reaction was insensitive to serine concentration in the range 2-20mM and was used to calculate kcat./Km values. However, kobs. was very sensitive to pH* values in the range 6.5-8.5 and the data require that the free enzyme is active in the base form resulting from two inseparable ionizations of pKa 7.3, and inactive after a third ionization controlled by a pKa of 7.5. Initial rates measured by u.v. absorbance and colorimetric procedures were used to calculate kinetic parameters of the tryptophan synthesis reaction. From pH 6.5 to 7, kcat./Km values for L-serine in the tryptophan synthesis and hydrogen exchange reactions were indistinguishable and increased rapidly under the control of two acid-base groups of pKa 6.7 and 7.2. Above pH 7, this equivalence breaks down because the exchange reaction alone is responsive to the third pKa value of the free enzyme. The pH dependence of the catalytic constant for tryptophan synthesis was qualitatively similar to that of the kobs. for serine exchange. A mechanism to explain the results is contrasted with recent proposals for the Escherichia coli system.

Limited proteolysis of the .beta.2-dimer of tryptophan synthase yields an enzymatically active derivative that binds .alpha.-subunits

Biochemistry ◽  
1991 ◽  
Vol 30 (17) ◽  
pp. 4173-4179 ◽  
Author(s):  
Michael Kaufmann ◽  
Thomas Schwarz ◽  
Rainer Jaenicke ◽  
Klaus D. Schnackerz ◽  
Helmut E. Meyer ◽  
...  
Keyword(s):  
Limited Proteolysis ◽  
Tryptophan Synthase ◽  
Active Derivative ◽  
Alpha Subunits ◽  
Beta 2

Preparation of Functionalized Diorganomagnesium Reagents in Toluene via Bromine or Iodine/Magnesium-Exchange Reactions

Synthesis ◽  
2021 ◽  
Author(s):  
Alexandre Desaintjean ◽  
Fanny Danton ◽  
Paul Knochel
Keyword(s):  
Exchange Reaction ◽  
Nitro Group ◽  
General Formula ◽  
Functional Groups ◽  
Exchange Reactions ◽  
Acyl Chlorides ◽  
Aryl Iodides ◽  
Highly Sensitive ◽  
Wide Range ◽  
Magnesium Exchange

A wide range of polyfunctionalized di(hetero)aryl- and dialkenyl-magnesium reagents were prepared in toluene within 10 to 120 min between −78 °C and 25 °C via an I/Mg- or Br/Mg-exchange reaction using reagents of the general formula R2Mg (R = sBu, Mes). Highly sensitive functional groups, such as a triazene or a nitro group, were tolerated in these exchange reactions, enabling the synthesis of various functionalized (hetero)arenes and alkenes derivatives after quenching with several electrophiles including allyl bromides, acyl chlorides, aldehydes, ketones, and aryl iodides.

scholarly journals Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Langxing Liao ◽  
Yonghui Zhang ◽  
Yali Wang ◽  
Yousi Fu ◽  
Aihui Zhang ◽  
...  
Keyword(s):  
Glucose Dehydrogenase ◽  
Catalytic Efficiency ◽  
Space Time ◽  
Cofactor Regeneration ◽  
Enzyme Complex ◽  
Regeneration System ◽  
Regeneration Rate ◽  
Leucine Dehydrogenase ◽  
Fusion Enzyme ◽  
Space Time Yield

Abstract Background Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. Results In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle.

THEORETICAL STUDIES ON THE COUPLING INTERACTIONS AND SELF-EXCHANGE REACTION MECHANISMS IN THE COMPLEXES OF NO WITH ONH AND NOH

2008 ◽  
Vol 07 (03) ◽  
pp. 435-446 ◽  
Author(s):  
PING LI ◽  
XIAOYAN XIE ◽  
YUXIANG BU ◽  
WEIHUA WANG ◽  
NANA WANG ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Exchange Reaction ◽  
Reaction Mechanisms ◽  
Natural Bond Orbital ◽  
Theoretical Studies ◽  
Exchange Reactions ◽  
Electron Transfer Mechanism ◽  
Proton Coupled Electron Transfer ◽  
Planar Geometries ◽  
Transfer Mechanisms

The coupling interactions and self-exchange reaction mechanisms between NO and ONH (NOH) have been systematically investigated at the B3LYP/6-311++G** level of theory. All the equilibrium complexes are characterized by the intermolecular H-bonds and co-planar geometries. The cisoid NOH/ON species is the most stable one among all the complexes considered due to the formation of an N – N bond. Moreover, all the cisoid complexes are found to be more stable than the corresponding transoid ones. The origin of the blueshifts occurring in the selected complexes has been explored, employing the natural bond orbital (NBO) calculations. Additionally, the proton transfer mechanisms for the self-exchange reactions have been proposed, i.e. they can proceed via the three-center proton-coupled electron transfer or five-center cyclic proton-coupled electron transfer mechanism.

scholarly journals Mitochondrial Thioredoxin-Glutathione Reductase from LarvalTaenia crassiceps(Cysticerci)

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Alberto Guevara-Flores ◽  
Irene P. del Arenal ◽  
Guillermo Mendoza-Hernández ◽  
Juan Pablo Pardo ◽  
Oscar Flores-Herrera ◽  
...  
Keyword(s):  
Glutathione Reductase ◽  
Reductase Activity ◽  
Catalytic Efficiency ◽  
Dependent Manner ◽  
Exchange Reactions ◽  
Disulfide Exchange ◽  
Oxidative Inactivation ◽  
Disulfide Reductase ◽  
Thioredoxin Peroxidase ◽  
Thioredoxin Glutathione Reductase

Mitochondrial thioredoxin-glutathione reductase was purified from larvalTaenia crassiceps(cysticerci). The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At25∘Cspecific activities were437  ±  27mU mg-1and840  ±  49mU mg-1with thioredoxin and GSSG, respectively. ApparentKmvalues were0.87  ±  0.04 μM,41  ±  6 μM and19  ±  10 μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

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