Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima.
D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Thermotoga maritima, a hyperthermophilic eubacterium, has been isolated in pure crystalline form. The enzyme is a homotetramer with a subunit molecular mass of 37 kDa. The sedimentation coefficient of the native enzyme is 7.3 X 10(-13)s, the isoelectric point is 4.6, and the specific absorption coefficient A1%, 1cm 280nm = 8.4. The enzyme shows extreme thermal stability: differential scanning calorimetry yields a transition temperature (Tm) of 109 degrees C for the NAD-saturated enzyme. Thermal deactivation occurs at T greater than 90 degrees C. The physicochemical characteristics of the enzyme suggest that its gross structure must be very similar to the structure of GAPDHs from mesophilic sources. The amino acid composition does not confirm the known "traffic rules" of thermal adaptation, apart from the Lys----Arg exchange. One reactive and at least two buried SH groups can be titrated with 5,5'-dithiobis(2-nitrobenzoate). The highly reactive SH group is probably the active-site cysteine residue common to all known GAPDHs. The activation energy of the glyceraldehyde 3-phosphate oxidation reaction decreases with increasing temperature. This functional behavior can be correlated with the temperature-dependent changes of both the intrinsic fluorescence and the near-UV circular dichroism; both indicate a temperature-dependent structural reorganization of the enzyme. Hydrogen-deuterium exchange reveals significantly increased rigidity of the thermophilic enzyme if compared to mesophilic GAPDHs at 25 degrees C, thus indicating that the conformational flexibility is similar at the corresponding physiological temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)
Wrba
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Institut für Biophysik und Physikalische Biochemie, Universität-Regensburg, FRG.
Schweiger
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A
Schultes
V
V
Jaenicke
R
R
Závodszky
P
P
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000596:Amino Acids; D005954:Glucosephosphate Dehydrogenase