title: Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase: structural basis for enhanced stability.
abstract: 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.
authors.affiliation: Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskie gory, Moscow 119991, Russia.
authors.forename: Yu L
authors.initials: YL
authors.lastname: Elkina
authors.forename: M L
authors.initials: ML
authors.lastname: Kuravsky
authors.forename: M A
authors.initials: MA
authors.lastname: El'darov
authors.forename: S V
authors.initials: SV
authors.lastname: Stogov
authors.forename: V I
authors.initials: VI
authors.lastname: Muronetz
authors.forename: E V
authors.initials: EV
authors.lastname: Schmalhausen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D011392:Proline; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases