Synthesis and kinetic evaluation of a trifunctional enzyme mimic with a dimanganese active centre

Gao, Nan; Li, Huanmin; Li, Quanshun; Luo, Gaoxing

doi:10.1016/j.jinorgbio.2010.09.014

Cited by 7 publications

(6 citation statements)

References 33 publications

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“…The slopes of the initial velocity rates were all similar, illustrating that the catalytic mechanism of Se‐CuZn‐65P is comparable with the mechanism of function of GPx. The K max /K H2O2 and K max /K GSH of the Se‐CuZn‐65P were calculated with the steady‐state dynamic equation, and they were 5.3 × 10 5 M −1 min −1 and 3.1 × 10 5 M −1 min −1 , respectively. Both values were 2 orders of magnitude lower than the values calculated for the natural GPx (K max /K H2O2 = 5.78 × 10 7 M −1 min −1 and K max /K GSH = 5.6 × 10 7 M −1 min −1 ).…”

Section: Resultsmentioning

confidence: 99%

“…The purified fusion protein PET32a(+)‐65P was digested by 4 U enterokinase for 18 hours in a buffer containing 25mM Tris‐HCl, pH = 7.6, 50mM NaCl, and 2mM CaCl 2 and then purified by HPLC. Se‐65P was prepared by chemical modification, as previously described . Ten milliliters of 65P solution were diluted with 10 mL of 10mM PBS (pH = 7), and 50 μL of PMSF solution (dissolved in 10 mg/mL acetonitrile) was added every 15 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…The kinetics data were obtained by double inverse mapping. The steady‐state kinetics equation for the reaction mechanism is as follows:

V_{0} / {[E]}_{0} = \frac{K_{\max} ([], GSH) ([], H_{2} O_{2})}{true(K_{H 2 O 2} ([], GSH) + K_{GSH} ([], H_{2} O_{2}) + ([], GSH) ([], H_{2} O_{2})} .

…”

Section: Methodsmentioning

confidence: 99%

“…The K max /K H2O2 and K max /K GSH of the Se-CuZn-65P were calculated with the steady-state dynamic equation, 8 Lipid peroxides are mitochondrial polyunsaturated fatty acids that produced by the ROS. Malondialdehyde is a characteristic product of lipid peroxides, which can be used to measure the degree of lipid peroxidation.…”

Section: The Mechanism Of Function Of the Se-cuzn-65pmentioning

confidence: 99%

“…Se-65P was prepared by chemical modification, as previously described. 8 Ten milliliters of 65P solution were diluted with 10 mL of 10mM PBS (pH = 7), and 50 μL of PMSF solution (dissolved in 10 mg/mL acetonitrile) was added every 15 minutes. The reaction was performed as previously described, 13 at room temperature for 3 hours; then nitrogen gas was piped in for 20 minutes, at which point 2 μL of NaHSe (1 mol/L) was added.…”

Section: The Preparation Of Se-cuzn-65pmentioning

confidence: 99%

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Study on a 65‐mer peptide mimetic enzyme with GPx and SOD dual function

Yin

Zhuang

et al. 2018

J of Molecular Recognition

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Excessive reactive oxygen species (ROS) levels are harmful to the body. The peroxidase, GPx, and the superoxide dismutase, SOD, are important antioxidant enzymes for preventing ROS-induced damage. Se-CuZn-65P is an enzyme mimetic with dual GPx and SOD antioxidant function. However, currently, its production is mainly based on the cysteine auxotrophic expression technique, which is inefficient, expensive, and time consuming. In this study, we combined protein engineering and the chemical mutation method to synthesize Se-CuZn-65P. The DNA sequence encoding the 65 amino acid peptide with the desired sequence transformations to incorporate the SOD and the GPx catalytic sites was cloned and expressed in a soluble protein expression vector. The protein yield increased up to 152 mg/L, which is 10 times higher than in previous studies. The SOD and GPx activity of Se-CuZn-65P was high (1181 U/mg and 753 U/μmol, respectively). The binding constant of glutathione was 5.6 × 10 L·mol , which shows that Se-CuZn-65P efficiently catalyzed hydrogen peroxide reduction by glutathione. Mitochondrial damage experiments confirmed the double protective role of the Se-CuZn-65P peptide and demonstrated functional synergy between the SOD and the GPx domains, which indicates its potential to be used in the treatment of ROS-related diseases. Our research may give a new thought to increase the yield of mimic.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The kinetics data were obtained by double inverse mapping. The steady‐state kinetics equation for the reaction mechanism is as follows: