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Post-translational Regulation of Hexokinase Function and Protein Stability in the Aestivating Frog Xenopus laevis

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Abstract

Xenopus laevis endure substantial dehydration which can impose hypoxic stress due to impaired blood flow. Tissues may increase reliance on anaerobic glycolysis for energy production making the regulation of hexokinase (HK) important. We investigated the enzymatic properties and phosphorylation state of purified HK from the muscle of control and dehydrated (30 % total body water lost) frogs. Bioinformatic tools were also applied to analyze the structural implication of HK phosphorylation in silico. HK from the muscle of dehydrated frogs showed a significantly higher Vmax (3.4-fold) and Km for glucose (2.4-fold) compared with control HK but the Km for ATP was unaltered. HK from dehydrated frogs also showed greater phosphoserine content (20 % increase) and lower phosphothreonine (22 % decrease) content compared to control HK. Control HK had a higher melting temperature (Tm = 61.9 °C) than from dehydrated (Tm = 54.2 °C) frogs when thermostability was tested using differential scanning fluorimetry. In silico phosphorylation of a Xenopus HK caused alterations in active site binding, corroborating phosphorylation as the probable mechanism for kinetic regulation. Physiological consequences of dehydration-induced HK phosphorylation appear to facilitate glycolytic metabolism in hypoxic situations. Augmented HK function increases the ability of Xenopus to overcome compromised oxidative phosphorylation associated with ischemia during dehydration.

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Abbreviations

DEAE:

Diethylaminoethyl

EGTA:

Ethylene glycol tetraacetic acid

EDTA:

Ethylenediaminetetraacetic acid

G6PDH:

Glucose-6-phosphate dehydrogenase

HK:

Hexokinase

LDH:

Lactate dehydrogenase

MOE:

Molecular operating environment

PMSF:

Phenylmethylsulfonyl fluoride

PPSP:

Prediction of PK-specific phosphorylation

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Acknowledgments

Thanks to JM Storey for editorial review of the manuscript, to Dr. Alex Wong for bioinformatics guidance and to Dr. Neal J. Dawson and Dr. Ryan A. V. Bell for generally teaching me everything. This research was supported by a discovery grant (#6793) from the Natural Sciences and Engineering Research Council (NSERC) of Canada to KBS and the Canada Research Chairs program.

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  1. Department of Biology, Institute of Biochemistry, Carleton University, Ottawa, ON, Canada

    Christine L. Childers & Kenneth B. Storey

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  1. Christine L. Childers
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  2. Kenneth B. Storey
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Correspondence to Kenneth B. Storey.

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Childers, C.L., Storey, K.B. Post-translational Regulation of Hexokinase Function and Protein Stability in the Aestivating Frog Xenopus laevis . Protein J 35, 61–71 (2016). https://doi.org/10.1007/s10930-016-9647-0

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