Conformational studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU.

Among the various proposals that have been made in attempting to explain the ability of thermophiles to reproduce at high temperatures, there is no doubt that obligate and extreme thermophiles synthesize proteins (and other molecules) that have sufficient intrinsic molecular stability to withstand increased thermal stress. In contrast, the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU has been shown to be quite thermolabile in vitro. Thermal inactivation is not due to loss of bound NAD+. It has also been shown that the enzymatic activity can be thermostabilized in vitro by increased ionic strength. As previously reported [J. W. Crabb, A. L. Murdock, and R. E. Amelunxen (1975) Biochem. Biophys. Res. Commun. 62, 627; (1977) Biochemistry 16, 4840], the enzyme loses 94-97% of enzymatic activity after heat treatment at 55 degrees C for 5 min in 0.05 M sodium phosphate buffer (pH 7.1); however, by increasing the ionic strength to 1.8, complete protection was conferred at this temperature. Gel-filtration chromatography has been used to study the initial dissociation and subsequent aggregation of the glyceraldehyde-3-phosphate dehydrogenase after thermal inactivation. Aggregation occurs when the enzyme is heated at 50 degrees or 55 degrees C. Loss of enzymatic activity is correlated with changes in the tertiary structure as measured by the near-uv CD spectrum of the enzyme following heat inactivation, with essential disappearance of the peaks at 263 and 296 nm, and a blue shift of the far-uv spectrum, which is a measure of secondary structure. Estimation of secondary structure of the unheated protein from the far-uv CD data showed the enzyme contains approximately 26% alpha-helix, approximately 21% beta-structure, and approximately 53% disordered structure. Heat treatment at various temperatures resulted in only slight changes of the estimated secondary structure. Increased ionic strength prevents thermal alteration of the CD spectrum in both near- and far-uv regions. The data support the previous proposal that thermolabile enzymes such as the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile B. coagulans are thermostabilized in vivo mainly by the intracellular charged macromolecular environment.

Molecular symmetry of glyceraldehyde-3-phosphate dehydrogenase from Bacillus coagulans.

The thermolabile glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans has a crystallographically exact 2-fold rotation axis of symmetry in one of its orthorhombic crystal forms (Lee et al., 1982). Using various crystallographic techniques, we have now identified this axis to be the molecular R-axis, which is the symmetry axis that relates the two subunits that form each active site of the tetrameric enzyme. This is in contrast to the symmetry of the human skeletal muscle enzyme wherein the crystallographically exact axis was found to be the Q-axis (Buehner et al., 1974). This finding could have important implications for the possible mechanism for the allosteric behavior of this molecule.

Sequence homology in the amino-terminal and active-site regions of thermolabile glyceraldehyde-3-phosphate dehydrogenase from a thermophile.

The unusual thermolability of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU (Crabb et al., Biochemistry 16:4840-4847, 1977) has provided the first opportunity to study a homologous enzyme from the same genus that exhibits a marked difference in thermostability. In pursuit of the structural bases for the thermostability of proteins, the sequences of the amino terminus (residues 1 through 27) and the active-site cysteine cyanogen bromide peptide (residues 130 through 167) of this enzyme have been determined and compared with sequences of the enzyme from other sources. The importance of comparing phylogenetically related proteins is evident from the 87% identity found between these sequences in the enzyme from B. coagulans and Bacillus stearothermophilus, versus only 45% identity for all other known sequences. The marked sequence identity of the enzyme from the two Bacillus species drew attention to the variable region (residues 138 through 140a) which is exposed to the exterior of the quaternary structure of this enzyme. Based on the reported crystallographic structures of the enzyme from lobster muscle and B. stearothermophilus and space-filling models of the variable region, the segment Asp-Pro-Lys-Ala in B. stearothermophilus should be more thermostable than the analogous sequence, Asp-Ala-Ala-Asn, from B. coagulans. In addition, the space-filling models suggested that the spatial relationship of an amino acid side chain and its potential for close packing and interactions with neighboring side chains may be more important than the type of amino acid substituted.