Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase: Structural basis for enhanced stability
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- 作者：Yu. L. Elkina ; M.L. Kuravsky ; M.A. El'darov ; S.V. Stogov ; V.I. Muronetz ; E.V. Schmalhausen
- 关键词：Sperm-specific glyceraldehyde-3-phosphate dehydrogenase ; GAPDS ; Spermatozoa ; Protein stability ; Proline rule
- 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
- 出版年：2010
- 出版时间：December 2010
- 年：2010
- 卷：1804
- 期：12
- 页码：2207-2212
- 全文大小：698 K
文摘
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 °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.