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Purification and properties of the first-identified, archaeal, ATP-dependent 6-phosphofructokinase, an extremely thermophilic non-allosteric enzyme, from the hyperthermophile Desulfurococcus amylolyticus

Purification and properties of the first-identified, archaeal, ATP-dependent... The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Desulfurococcus amylolyticus was purified 1500-fold to homogeneity. The enzyme had an apparent molecular mass of 140 kDa and was composed of a single type of subunit of 33 kDa suggesting a homotetrameric (α4) structure. The N-terminal amino acid sequence did not show significant similarity to ATP-PFKs isolated from eubacteria and eukarya. Kinetic constants of the enzyme were determined for both reaction directions at pH 6 and at 85 °C. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent K ms for ATP and fructose 6-phosphate (forward reaction) were 0.28 and 1.17 mM, respectively; the apparent V max was about 41 U/mg. ATP could not be replaced by pyrophosphate (PPi) or ADP as phosphoryl donor, thus defining the enzyme as an ATP-dependent PFK. In addition to ATP (100%), the enzyme accepted GTP (97%), ITP (130%), UTP (84%), CTP (55%) and, less effectively, acetyl phosphate (13%) as phosphoryl donors. Enzyme activity was not allosterically regulated by classical effectors of ATP-PFKs such as ADP, AMP, and phosphoenolpyruvate or citrate. The enzyme also catalysed in vitro the reverse reaction with an apparent K m for fructose-1,6-bisphosphate and ADP of 16.7 and 0.5 mM, respectively, and an apparent V max of about 4.5 U/mg. Divalent cations were required for maximal activity; Mg2+, which was most effective, could be replaced partially by Ni2+, Mn2+ or Co2+. The enzyme had a temperature optimum of 90 °C and showed a significant thermostability up to 100 °C, which is in accordance with its physiological function under hyperthermophilic conditions. This is the first description of an ATP-dependent PFK from the domain of archaea, characterized as an extremely thermophilic, non-allosteric enzyme. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Microbiology Springer Journals

Purification and properties of the first-identified, archaeal, ATP-dependent 6-phosphofructokinase, an extremely thermophilic non-allosteric enzyme, from the hyperthermophile Desulfurococcus amylolyticus

Hansen, Thomas; Schönheit, Peter
Archives of Microbiology , Volume 173 (2) – Nov 30, 1999
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References (21)

Publisher
Springer Journals
Copyright
Copyright © 1999 by Springer-Verlag
Subject
Life Sciences; Microbiology; Microbial Ecology; Biochemistry, general; Cell Biology; Biotechnology; Ecology
ISSN
0302-8933
eISSN
1432-072X
DOI
10.1007/s002039900114
Publisher site
See Article on Publisher Site

Abstract

The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Desulfurococcus amylolyticus was purified 1500-fold to homogeneity. The enzyme had an apparent molecular mass of 140 kDa and was composed of a single type of subunit of 33 kDa suggesting a homotetrameric (α4) structure. The N-terminal amino acid sequence did not show significant similarity to ATP-PFKs isolated from eubacteria and eukarya. Kinetic constants of the enzyme were determined for both reaction directions at pH 6 and at 85 °C. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent K ms for ATP and fructose 6-phosphate (forward reaction) were 0.28 and 1.17 mM, respectively; the apparent V max was about 41 U/mg. ATP could not be replaced by pyrophosphate (PPi) or ADP as phosphoryl donor, thus defining the enzyme as an ATP-dependent PFK. In addition to ATP (100%), the enzyme accepted GTP (97%), ITP (130%), UTP (84%), CTP (55%) and, less effectively, acetyl phosphate (13%) as phosphoryl donors. Enzyme activity was not allosterically regulated by classical effectors of ATP-PFKs such as ADP, AMP, and phosphoenolpyruvate or citrate. The enzyme also catalysed in vitro the reverse reaction with an apparent K m for fructose-1,6-bisphosphate and ADP of 16.7 and 0.5 mM, respectively, and an apparent V max of about 4.5 U/mg. Divalent cations were required for maximal activity; Mg2+, which was most effective, could be replaced partially by Ni2+, Mn2+ or Co2+. The enzyme had a temperature optimum of 90 °C and showed a significant thermostability up to 100 °C, which is in accordance with its physiological function under hyperthermophilic conditions. This is the first description of an ATP-dependent PFK from the domain of archaea, characterized as an extremely thermophilic, non-allosteric enzyme.

Journal

Archives of Microbiology – Springer Journals

Published: Nov 30, 1999

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