Glyceraldehyde 3-Phosphate Dehydrogenase of Bacillus stearothermophilusKinetics and Physicochemical Studies

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Abstract
The structure of glyceraldehyde 3-phosphate dehydrogenase [EC 1. 2.1.12] of B. stearothermophilus was investigated by circular dichroism and fluorescence emission spectro-scopy under various conditions. Spectrophotometric titration of tyrosine residues, and some kinetic studies were also performed. 1. The gross structure of the thermophilic enzyme was very similar to those of enzymes from other sources as judged by CD spectra. Thus, the thermophilic enzyme probably has a high content of β-structure. In the thermophilic enzyme many tyrosine residues showed a higher pK value (11.8) of the phenolic hydroxyl group, and tryptophan residues were buried in a more hydrophobic region as compared to the rabbit enzyme. These results indicate that hydrophobic interactions, may be one of the mechanisms for stabilization of the enzyme structure. 2. Km values for NAD+ and glyceraldehyde 3-phosphate did not change significantly with temperature, though the temperature coefficient (Q10) of Vmax was about 1.7. 3. The structure of the enzyme did not change in buffer up to the temperature (ca. 60°C) at which thermal inactivation occurred. In SDS no obvious structural change of the enzyme was detected under conditions where most oligomeric proteins-dissociate into subunits. 4. The enzyme gradually unfolded in urea solution. The unfolding was accelerated by heating, but rapid refolding of the polypeptide chain was observed when the urea was diluted at room temperature. 5. The enzyme showed a biphasic thermal inactivation curve in intermediate concentrations of urea (2–5 M). In lower (0–1 m) or higher (7 M) concentrations of urea the enzyme showed a monophasic inactivation curve. 6. A model for the denaturation and renaturation of the enzyme was proposed on the basis of the results obtained. In the model a rigid core of the enzyme, which may correspond to the active center region, is introduced. Some other discussions are made regarding the structure and activity of the enzyme.

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