Thermostable aldolase from Thermus aquaticus.
Data are presented on the purification and properties of the thermostable fructose-1,6-diphosphate aldolase of Thermus aquaticus, a nonsporulating, extreme thermophile. The enzyme shows little activity at temperatures below 60 C and optimal activity at about 95 C. The enzyme was purified 43-fold by diethylaminoethyl cellulose column chromatography and Sephadex G-200 gel filtration. The enzyme is activated by high concentrations of NH(4) (+) and low concentrations of Fe(2+) and Co(2+) and is strongly inhibited by ethylenediaminetetraacetic acid (EDTA). The activation by Fe(2+) and Co(2+) and the inhibition by EDTA are both reversed by dialysis. The enzyme is greatly activated by cysteine and less so by other sulfhydryl compounds. Activation by cysteine is reversible by dialysis. The purified enzyme had a molecular weight as determined by Sephadex G-200 gel filtration of 140,000; after incubation of enzyme with cysteine, another molecular species was also found with a molecular weight of 70,000. The purified enzyme is stable at low protein concentrations to 97 C but is rapidly inactivated at 105 C. In cysteine the enzyme is more heat labile; heat inactivation in the presence of cysteine is prevented by substrate, although, in the absence of cysteine, substrate partially labilizes the enzyme to heat. The temperature optimum for enzyme activity is several degrees lower in the presence of cysteine than in its absence, and the K(m) is threefold lower. It is concluded that the T. aquaticus enzyme resembles some other aldolases of Rutter's class II, except for its extreme heat stability. The T. aquaticus enzyme is compared with that of Bacillus stearothermophilus, a moderate thermophile. Although the T. aquaticus enzyme is considerably more heat stable, the enzymes from the two thermophiles have many similarities. New data are presented which show that the B. stearothermophilus aldolase is metal ion-dependent, in disagreement with earlier reports.
D001407:Bacillus / Q000201:enzymology; D001419:Bacteria / Q000032:analysis / Q000201:enzymology* / Q000254:growth & development; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003035:Cobalt / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D003956:Dialysis; D004492:Edetic Acid / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology; D008970:Molecular Weight; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D000644:Quaternary Ammonium Compounds; D013438:Sulfhydryl Compounds; D003035:Cobalt; D004492:Edetic Acid; D007501:Iron; D005634:Fructose-Bisphosphate Aldolase; D003545:Cysteine