Thermostable aldolase from Thermus aquaticus

Area of Science:

• Biochemistry
• Enzymology
• Extremophile Biology

Background:

• Fructose-1,6-diphosphate aldolase is a key enzyme in glycolysis.
• Thermostable enzymes from extremophiles offer insights into protein stability and function at high temperatures.

Purpose of the Study:

• To purify and characterize the fructose-1,6-diphosphate aldolase from the extreme thermophile Thermus aquaticus.
• To investigate the enzyme's properties, including optimal temperature, cofactor requirements, and stability.
• To compare the enzyme with aldolases from other thermophilic organisms.

Main Methods:

• Purification using diethylaminoethyl cellulose chromatography and Sephadex G-200 gel filtration.
• Enzyme activity assays at various temperatures and in the presence of different activators/inhibitors.
• Molecular weight determination via gel filtration.
• Heat inactivation studies.

Main Results:

• The enzyme was purified 43-fold and shows optimal activity around 95°C.
• It is activated by NH4+, Fe2+, Co2+, and cysteine, and inhibited by EDTA.
• Molecular weight was determined to be 140,000, with a 70,000 species appearing after cysteine incubation.
• The enzyme is highly heat-stable but becomes more heat labile in the presence of cysteine, though substrate protects against heat inactivation.

Conclusions:

• The Thermus aquaticus aldolase shares characteristics with Rutter's class II aldolases but possesses extreme heat stability.
• It is significantly more heat-stable than the aldolase from Bacillus stearothermophilus.
• New data suggest Bacillus stearothermophilus aldolase is metal ion-dependent, contrary to previous findings.