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Biochim Biophys Acta. 2010 Dec;1804(12):2207-12. PubMed; doi 10.1016/j.bbapap.2010.09.002

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase: structural basis for enhanced stability.

Elkina, Y. L.; Kuravsky, M. L.; El'darov, M. A.; Stogov, S. V.; Muronetz, V. I.; Schmalhausen, E. V.

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is bound to the fibrous sheath of the sperm flagellum through the hydrophobic N-terminal domain of the enzyme molecule. Expression of human GAPDS in E.coli cells yields inactive and insoluble protein. Presumably, the N-terminal domain prevents correct folding of the full-length recombinant enzyme. To obtain GAPDS in a soluble and active form, a recombinant enzyme lacking in 68 amino acids of the N-terminal domain (dN-GAPDS) was expressed in E.coli cells. Purified dN-GAPDS was shown to be a protein of 9.3 nm in diameter (by dynamic light scattering), which is close to the size of the muscle tetrameric glyceraldehyde-3-phosphate dehydrogenase (8.6 nm). The catalytic properties of the protein differed a little from those of the muscle glyceraldehyde-3-phoshate dehydrogenase. However, compared to muscle glyceraldehyde-3-phoshate dehydrogenase, dN-GAPDS exhibited enhanced thermostability (the transition midpoints values are 60.8 and 67.4 degrees C, respectively) and was much more resistant towards action of guanidine hydrochloride (inactivation constants are 2.45+/-0.018 and 0.118 +/- 0.008 min(-1), respectively). The enhanced stability of dN-GAPDS is likely to be related to some specific features of the GAPDS structure compared to that of the muscle enzyme: 1) reduced number of solvent-exposed salt bridges; 2) 2 additional buried salt bridges; and 3) 6 additional proline residues in GAPDS meeting the "proline rule". It is assumed that high stability of the sperm-specific GAPDS is of importance for the efficiency of fertilization.



 


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