scholarly journals Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

2002 ◽  
Vol 184 (15) ◽  
pp. 4096-4103 ◽  
Author(s):  
Yong Ge ◽  
Frédéric H. Vaillancourt ◽  
Nathalie Y. R. Agar ◽  
Lindsay D. Eltis
Keyword(s):  
Steady State ◽  
Pseudomonas Putida ◽  
Sodium Phosphate ◽  
Ternary Complex ◽  
Specific Activity ◽  
Complex Mechanism ◽  
Aromatic Substrate ◽  
Steady State Kinetics ◽  
Full Complement

ABSTRACT Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe2S2 centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, k cat = 3.9 ± 0.2 s−1, and K m O2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where K m O2 represents the K m for O2 and KmA represents the K m for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ≃ 3-chlorobenzoate > p-toluate ≃ 4-chlorobenzoate ≫ o-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate. However, the apparent K m of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates.

scholarly journals Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase

2005 ◽  
Vol 386 (2) ◽  
pp. 305-314 ◽  
Author(s):  
Edwin J. A. VELDHUIZEN ◽  
Frédéric H. VAILLANCOURT ◽  
Cheryl J. WHITING ◽  
Marvin M.-Y. HSIAO ◽  
Geneviève GINGRAS ◽  
...  
Keyword(s):  
Steady State ◽  
Active Site ◽  
Ternary Complex ◽  
Specific Activity ◽  
Complex Mechanism ◽  
Substrate Analogues ◽  
Steady State Kinetics ◽  
Km Value ◽  
Substrate Concentrations ◽  
Active Preparation

HGO (homogentisate 1,2-dioxygenase; EC 1.13.11.5) catalyses the O2-dependent cleavage of HGA (homogentisate) to maleylacetoacetate in the catabolism of tyrosine. Anaerobic purification of heterologously expressed Fe(II)-containing human HGO yielded an enzyme preparation with a specific activity of 28.3± 0.6 μmol·min−1·mg−1 (20 mM Mes, 80 mM NaCl, pH 6.2, 25 °C), which is almost twice that of the most active preparation described to date. Moreover, the addition of reducing agents or other additives did not increase the specific activity, in contrast with previous reports. The apparent specificity of HGO for HGA was highest at pH 6.2 and the steady-state cleavage of HGA fit a compulsory-order ternary-complex mechanism (Km value of 28.6±6.2 μM for HGA, Km value of 1240±160 μM for O2). Free HGO was subject to inactivation in the presence of O2 and during the steady-state cleavage of HGA. Both cases involved the oxidation of the active site Fe(II). 3-Cl HGA, a potential inhibitor of HGO, and its isosteric analogue, 3-Me HGO, were synthesized. At saturating substrate concentrations, HGO cleaved 3-Me and 3-Cl HGA 10 and 100 times slower than HGA respectively. The apparent specificity of HGO for HGA was approx. two orders of magnitude higher than for either 3-Me or 3-Cl HGA. Interestingly, 3-Cl HGA inactivated HGO only twice as rapidly as HGA. This contrasts with what has been observed in mechanistically related dioxygenases, which are rapidly inactivated by chlorinated substrate analogues, such as 3-hydroxyanthranilate dioxygenase by 4-Cl 3-hydroxyanthranilate.

Transient-Phase and Steady-State Kinetics for Enzyme Systems Involving Two Substrates

1973 ◽  
Vol 51 (6) ◽  
pp. 832-840 ◽  
Author(s):  
Nasrat H. Hijazi ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Ternary Complex ◽  
Transient Phase ◽  
Complex Mechanism ◽  
State Equations ◽  
Enzyme Systems ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
The Individual ◽  
Individual Rate

The transient-phase and steady-state equations are derived for four enzyme mechanisms involving two substrates, namely (1) Theorell–Chance mechanism, (2) ping pong bi bi mechanism, (3) ordered ternary-complex mechanism, and (4) random ternary-complex mechanism. In each case, a discussion is presented of the way in which the individual rate constants can be separated on the basis of experimental transient-phase investigations.

scholarly journals Dissociation and catalysis in yeast hexokinase A.

1976 ◽  
Vol 155 (3) ◽  
pp. 661-667 ◽  
Author(s):  
D C Williams ◽  
J G Jones
Keyword(s):  
Catalytic Activity ◽  
Steady State ◽  
Ternary Complex ◽  
Specific Activity ◽  
Temperature Dependent ◽  
Gradual Decline ◽  
Experimental Conditions ◽  
Progress Curve ◽  
Catalytically Active ◽  
Yeast Hexokinase

1. The specific activity of yeast hexokinase A depends on the concentration of the protein in the solution being assayed. When a solution containing 13.5 mg of hexokinase A/ml is diluted 10-100-fold at various values of pH and temperature, there is a gradual decline in the specific activity of the enzyme until an equilibrium value is reached, which varies with the chosen experimental conditions. 2. The catalytic activity lost when hexokinase A (1 mg/ml) is incubated at 30degreesC is recovered by lowering the temperature to 25degreesC. 3. These concentration- and temperature-dependent phenomena are consistent with the existence of a monomer-dimer equilibrium in which the dimer alone is the catalytic form of the enzyme. 4. Glucose alone prevents the decline in specific activity of hexokinase A after dilution, but it does not re-activate dilute solutions solutions of the enzyme. It is concluded that glucose binds to both the dimer and the monomer and prevents both association and dissociation. 5. The progress curve describing the phosphorylation of glucose catalysed by hexokinase A does not attain a steady state. It is possible that dissociation of catalytically active dimers in a ternary complex with glucose and ATP (or glucose 6-phosphate and ADP) could explain the non-linearity of this progress curve.

scholarly journals Bovine lens aldehyde dehydrogenase. Kinetics and mechanism

1983 ◽  
Vol 215 (2) ◽  
pp. 361-368 ◽  
Author(s):  
H H Ting ◽  
M J C Crabbe
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Ternary Complex ◽  
Succinic Semialdehyde ◽  
Complex Mechanism ◽  
Thiol Groups ◽  
Bovine Lens ◽  
Steady State Kinetic ◽  
State Kinetic

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.

scholarly journals Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 415 ◽  
Author(s):  
Haitao Ding ◽  
Lili Zhou ◽  
Qian Zeng ◽  
Yong Yu ◽  
Bo Chen
Keyword(s):  
Steady State ◽  
Heterologous Expression ◽  
Specific Activity ◽  
Recombinant Enzyme ◽  
Size Exclusion ◽  
Wild Type ◽  
Promising Alternative ◽  
Steady State Kinetics ◽  
Wild Type Counterpart ◽  
Exclusion Chromatography

A thermostable β-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD600 of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme β-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg−1 at 37 °C using ONPG (o-nitrophenyl-β-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3′-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with β-1,4 linkage and β-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with β-1,3 linkage.

Characterization of the PLP-dependent transaminase initiating azasugar biosynthesis

2018 ◽  
Vol 475 (13) ◽  
pp. 2241-2256
Author(s):  
Jeffrey M. Arciola ◽  
Nicole A. Horenstein
Keyword(s):  
Steady State ◽  
Specific Activity ◽  
Paenibacillus Polymyxa ◽  
Binding Pocket ◽  
Kinetic Mechanism ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Amino Donor ◽  
Event Based

Biosynthesis of the azasugar 1-deoxynojirimycin (DNJ) critically involves a transamination in the first committed step. Here, we identify the azasugar biosynthetic cluster signature in Paenibacillus polymyxa SC2 (Ppo), homologous to that reported in Bacillus amyloliquefaciens FZB42 (Bam), and report the characterization of the aminotransferase GabT1 (named from Bam). GabT1 from Ppo exhibits a specific activity of 4.9 nmol/min/mg at 30°C (pH 7.5), a somewhat promiscuous amino donor selectivity, and curvilinear steady-state kinetics that do not reflect the predicted ping-pong behavior typical of aminotransferases. Analysis of the first half reaction with l-glutamate in the absence of the acceptor fructose 6-phosphate revealed that it was capable of catalyzing multiple turnovers of glutamate. Kinetic modeling of steady-state initial velocity data was consistent with a novel hybrid branching kinetic mechanism which included dissociation of PMP after the first half reaction to generate the apoenzyme which could bind PLP for another catalytic deamination event. Based on comparative sequence analyses, we identified an uncommon His-Val dyad in the PLP-binding pocket which we hypothesized was responsible for the unusual kinetics. Restoration of the conserved PLP-binding site motif via the mutant H119F restored classic ping-pong kinetic behavior.

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