The investigation of regulatory transition in D‐glyceraldehyde‐3‐phosphate dehydrogenase quaternary structure

Markovich, Daniel; Krapivinskaya, D.

doi:10.1016/0014-5793(75)80243-2

Cited by 5 publications

(2 citation statements)

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“…Active heterotetramers of GAPDH between different species have been reported and biochemically characterized previously, both in ratios of 2:2 and 3:1 (37)(38)(39)(40). In this study we have successfully obtained crystals of rat recombinant GAPDS as a heterotetramer with Escherichia coli GAPDH in a 1:3 ratio.…”

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confidence: 74%

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Frayne

Taylor

Cameron

et al. 2009

Journal of Biological Chemistry

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Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein.A structure was also obtained where glyceraldehyde 3-phosphate binds in the P s pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of ␣-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.Glyceraldehyde-3-phosphate dehydrogenase-S (GAPDS 3 in rat; GAPDH2 in human) is the sperm-specific isoform of GAPDH (1-3) and the sole GAPDH enzyme in sperm. GAPDS is highly conserved between species showing 94% identity between rat and mouse and 87% identity between rat and human. Within a particular species, GAPDS also shows significant sequence identity to its GAPDH paralogue, 70, 70, and 68% for rat, mouse, and human, respectively. The most striking difference between GAPDS and GAPDH is the presence of an N-terminal polyproline region in GAPDS, which is 97 residues in rat (accession number AJ297631), 105 in mouse (3), and 72 in human (2). GAPDS is restricted to the principal piece of the sperm flagellum (1, 2, 4) where it is localized to the fibrous sheath (5), an association proposed to be mediated via the N-terminal polyproline extension.GAPDS first came to prominence as a contraceptive target during the 1970s (6 -8). Investigations showed that treatment of sperm with ␣-chlorohydrin or a number of related compounds could inhibit GAPDS activity (9 -11), sperm motility (9 -13), and the fertilization of oocytes in vitro (14). The metabolite of these compounds, 3-chlorolactaldehyde (15-17), selectively inhibited GAPDS, having no effect on the activity of somatic cell GAPDH (18,19), pro...

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confidence: 74%

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Frayne

Taylor

Cameron

et al. 2009

Journal of Biological Chemistry

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“…29,30 Monoand di-mercurized products were fractionated by gel filtration through a Sephadex G-50 column (5 x 50 cm; Phamacia, Uppsala, Sweden) in 0.01 M NaOH. Fluoresceine monomercury acetate (FMMA) is an efficient and highly specific reagent for Cys residues of proteins, forming a covalent bond with SH groups.…”

Section: Synthesis and Spectral Characteristics Of Fmmamentioning

confidence: 99%

Specific fluorescent labeling of two functional domains in RNA polymerase α subunit

et al. 1998

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A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the alpha. subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated alpha subunit and alpha subunit assembled in the holoenzyme. In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements. Substitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of alpha subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for alpha dimerization and its binding of beta' subunit. In the isolated alpha subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant alpha-subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes. This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit.

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Photosynthesis. Biochemical and Physiological Aspects of Carbon Metabolism

Latzko

Kelly

Thirty Years of Photosynthesis 1974–2004

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The investigation of regulatory transition in D‐glyceraldehyde‐3‐phosphate dehydrogenase quaternary structure

Cited by 5 publications

References 9 publications

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Specific fluorescent labeling of two functional domains in RNA polymerase α subunit

Photosynthesis. Biochemical and Physiological Aspects of Carbon Metabolism

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