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Binding of a burst-phase intermediate formed in the folding of denatured D-glyceraldehyde-3-phosphate dehydrogenase by chaperonin 60 and 8-anilino-1-naphthalenesulphonic acid.

ABSTRACT: Upon dilution, D-glyceraldehyde-3-phosphate dehydrogenase (GADPH) that has been fully inactivated, but only partially unfolded, in dilute guanidine hydrochloride (GuHCl) recovers activity completely. The fully unfolded enzyme, however, is re-activated only to a limited extent after dilution, and refolds rapidly in a burst phase to a partially folded intermediate characterized by increases in both the emission intensity of intrinsic fluorescence and binding to 8-anilino-1-naphthalenesulphonic acid (ANS). This intermediate aggregates with a time lag of a few minutes, and the aggregation can be suppressed completely by chaperonin 60 (GroEL). Stoichiometric analysis of the suppression of GAPDH re-activation by GroEL suggests that the tetradecameric GroEL binds to a dimeric GAPDH folding intermediate. This intermediate can be re-activated by ATP or ATP/chaperonin 10 (GroES) to an extent considerably greater than that obtained on spontaneous re-activation of the fully denatured enzyme upon dilution. Probing with a fluorescent derivative of NAD+ shows that this folding intermediate does not have a native conformation at the active site. The similar profiles of the effects of GroEL and ANS on the re-activation of GAPDH denatured by different concentrations of GuHCl suggest that GroEL and ANS recognize and bind to the same folding intermediate, which is similar to the relatively stable, partially unfolded, state of the enzyme denatured in 0.5-1.0 MGuHCl. However, the complexes of the intermediate with GroEL or ANS appear to be different, in that GroEL, but not ANS, suppresses aggregation and assists folding in the presence of ATP.

SUBMITTER: Li XL

PROVIDER: S-EPMC1219382 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

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Project description:1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these 'surface' amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.

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Binding of a burst-phase intermediate formed in the folding of denatured D-glyceraldehyde-3-phosphate dehydrogenase by chaperonin 60 and 8-anilino-1-naphthalenesulphonic acid.

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