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Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

Fang B, Niu J, Ren H, Guo Y, Wang S - PLoS ONE (2014)

Bottom Line: By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation.The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively.A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China.

ABSTRACT
Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

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Comparison of the simulated values with the experimental data for GDH.reaction conditions: NAD+ (□: 1.0 mmol/L; Δ: 0.8 mmol/L, Ο: 0.4 mmol/L, +: 0.2 mmol/L, ×: 0.12 mmol/L), simulated (lines, —); glycerol, 0.1 mol/L; GDH, 1 mg/L; pH, 12.0; temperature, 45°C.
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pone-0099162-g006: Comparison of the simulated values with the experimental data for GDH.reaction conditions: NAD+ (□: 1.0 mmol/L; Δ: 0.8 mmol/L, Ο: 0.4 mmol/L, +: 0.2 mmol/L, ×: 0.12 mmol/L), simulated (lines, —); glycerol, 0.1 mol/L; GDH, 1 mg/L; pH, 12.0; temperature, 45°C.

Mentions: The values of the kinetic parameters, namely KmA, KmB, KiA, KiB, KiP, KiQ, was obtained previously, which were used for the final parameter estimation via nonlinear regression [26]. Keq is a dependent parameter. Therefore, the number of estimated parameters was reduced from 10 to 4. Kinetic parameters for estimation can be remarkably decreased. The concentration-time curve data with various substrate concentrations were used for the simulation of the remaining four parameters, namely, Vf, Vr, KmP, and KmQ, by the MATLAB program. Parameter estimation was carried out by a combination of fourth- and fifth-order Runge kutta method using ode45 module in MATLAB software. All datasets were fitted at once. These parameters are listed in Table 2. The comparisons of simulated values with the experimental data for Mn-GDH and GDH are shown in Fig. 5 and Fig. 6. There was a good agreement between the experimental and simulated values, with 11.5% and 12.7% average relative error for Mn-GDH and GDH, respectively.


Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

Fang B, Niu J, Ren H, Guo Y, Wang S - PLoS ONE (2014)

Comparison of the simulated values with the experimental data for GDH.reaction conditions: NAD+ (□: 1.0 mmol/L; Δ: 0.8 mmol/L, Ο: 0.4 mmol/L, +: 0.2 mmol/L, ×: 0.12 mmol/L), simulated (lines, —); glycerol, 0.1 mol/L; GDH, 1 mg/L; pH, 12.0; temperature, 45°C.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4045801&req=5

pone-0099162-g006: Comparison of the simulated values with the experimental data for GDH.reaction conditions: NAD+ (□: 1.0 mmol/L; Δ: 0.8 mmol/L, Ο: 0.4 mmol/L, +: 0.2 mmol/L, ×: 0.12 mmol/L), simulated (lines, —); glycerol, 0.1 mol/L; GDH, 1 mg/L; pH, 12.0; temperature, 45°C.
Mentions: The values of the kinetic parameters, namely KmA, KmB, KiA, KiB, KiP, KiQ, was obtained previously, which were used for the final parameter estimation via nonlinear regression [26]. Keq is a dependent parameter. Therefore, the number of estimated parameters was reduced from 10 to 4. Kinetic parameters for estimation can be remarkably decreased. The concentration-time curve data with various substrate concentrations were used for the simulation of the remaining four parameters, namely, Vf, Vr, KmP, and KmQ, by the MATLAB program. Parameter estimation was carried out by a combination of fourth- and fifth-order Runge kutta method using ode45 module in MATLAB software. All datasets were fitted at once. These parameters are listed in Table 2. The comparisons of simulated values with the experimental data for Mn-GDH and GDH are shown in Fig. 5 and Fig. 6. There was a good agreement between the experimental and simulated values, with 11.5% and 12.7% average relative error for Mn-GDH and GDH, respectively.

Bottom Line: By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation.The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively.A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, China.

ABSTRACT
Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

Show MeSH
Related in: MedlinePlus