Functional characterization of an extreme thermophilic class II fructose-1,6-bisphosphate aldolase.
Fructose-1,6-bisphosphate aldolase activity has been isolated and purified to homogeneity from the extreme thermophile eubacteria Thermus aquaticus. The homogeneous enzyme is a class II aldolase as fructose-1,6-bisphosphate cleavage activity was strongly inhibited by EDTA, and activated by Co2+ metal ion. Taq aldolase is a stable tetramer with estimated molecular mass of 165 kDa. The enzyme is thermostable and is not inactived after heating at 90 degrees C for 2 h but looses 80% of activity after 1 h at 97 degrees C. The pH profile corresponding to maximal aldolase activity is displaced to more acidic values compared to other class II aldolases. Enzyme activation by both detergents and alcohols and chromatographic behaviour on hydrophobic stationary phases is consistent with presence of hydrophobic surface regions on the soluble enzyme. Kinetic behaviour of T. aquaticus aldolase at high fructose-1,6-bisphosphate concentrations indicates significant negative cooperativity. The Taq aldolase NH2-terminal sequence was determined and compared with available sequences from other class II aldolases. Significant sequence similarity was found between Taq aldolase and the thermostable aldolase from Bacillus stearothermophilus.
Department of Biochemistry, Université de Montréal, Canada.
C
C
De Montigny
J
J
Sygusch
D000438:Alcohols / Q000494:pharmacology; D000595:Amino Acid Sequence; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D005634:Fructose-Bisphosphate Aldolase / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D016415:Sequence Alignment; D017421:Sequence Analysis; D013696:Temperature; D013824:Thermus / Q000201:enzymology*
D000438:Alcohols; D003902:Detergents; D008670:Metals; D005634:Fructose-Bisphosphate Aldolase