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A Numerical Approach for Kinetic Analysis of the Nonexponential Thermoinactivation Process of Uricase

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Abstract

Prior to the exponential decrease of activity of a uricase from Candida sp. during storage at 37 °C, there was a plateau period of about 4 days at pH 7.4, 12 days at pH 9.2, and about 22 days in the presence of 30 μM oxonate at pH 7.4 or 9.2, but no degradation of polypeptides and no activity of resolved homodimers. To reveal determinants of the plateau period, a dissociation model involving a serial of conformation intermediates of homotetramer were proposed for kinetic analysis of the thermoinactivation process. In the dissociation model, the roles of interior noncovalent interactions essential for homotetramer integrity were reflected by an equivalent number of the artificial weakest noncovalent interaction; to avoid covariance among parameters, the rate constant for disrupting the artificial weakest noncovalent interaction was fixed at the minimum for physical significance of other parameters; among thermoinactivation curves simulated by numerical integration with different sets of parameters, the one for least-squares fitting to an experimental one gave the solution. Results found that the equivalent number of the artificial weakest noncovalent interaction primarily determined the plateau period; kinetics rather than thermodynamics for homotetramer dissociation determined the thermoinactivation process. These findings facilitated designing thermostable uricase mutants.

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Abbreviations

DD:

The denatured homodimer

H:

The nondenatured homodimer

k 1 :

The disruption rate of the weakest interaction

k −1 :

Reversal formation rate

k −2 :

The rate for the association of two nondenatured homodimers into the homotetramer

k 2 :

The rate for the denaturation of homodimer

LSF:

Least-squares fitting

m :

The equivalent number of the artificial weakest noncovalent interaction

PAGE:

Polyacrylamide electrophoresis

SDS-PAGE:

Sodium-dodecylsulfate polyacrylamide-electrophoresis

T1 :

The dynamic level of the homotetramer tolerating no disruption of interior noncovalent interactions essential for homotetramer integrity

Tm :

The dynamic level of representative conformational intermediate of the homotetramer (whose subscript indicated the conformational intermediate after the disruption of the mth interior noncovalent interaction in the dissociation pathway)

TA:

The total activity

T0.5 :

The temperature for reserving half of initial activity of an enzyme after the treatment for a customized time

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Acknowledgments

The work was supported by National Natural Science Foundation of China (Nos. 30672139, 31570862, 21402108), Sciences and Technology Commission of Yuzhong District of Chongqing (No. 20130135) and Education Ministry of China (No. 20125503110007). Miss Xin Wang prepared samples for Tandem MS–MS and Edman degradation analyses. The mutants of Bacillus fastidiosus uricase bearing plateau periods were already included in an invention patent application in China (Application No. 201610093491.8).

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    Authors and Affiliations

    1. Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China

      Jing Wu, Xiaolan Yang, Deqiang Wang, Xiaolei Hu, JingJing Rao, Jun Pu & Fei Liao

    2. Central Laboratory, Yongchuan Hospital, Chongqing Medical University, No.439, Xuanhua Road, Yongchuan, Chongqing, 402160, China

      Juan Liao

    3. Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA

      Chang-guo Zhan

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    Jing Wu and Xiaolan Yang have contributed equally to this work.

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    Wu, J., Yang, X., Wang, D. et al. A Numerical Approach for Kinetic Analysis of the Nonexponential Thermoinactivation Process of Uricase. Protein J 35, 318–329 (2016). https://doi.org/10.1007/s10930-016-9675-9

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