@PhdThesis{duepublico_mods_00026846,
  author = 	{Haferkamp, Patrick},
  title = 	{Biochemical studies of enzymes involved in glycolysis of the thermoacidophilic crenarchaeon Sulfolobus solfataricus},
  year = 	{2011},
  month = 	{Dec},
  day = 	{05},
  abstract = 	{Within the Sulfolobus Systems Biology (``SulfoSYS'') project the effect of temperature on a metabolic network is investigated at transcriptomic, metabolomic, proteomic and enzyme level. S. solfataricus utilizes an unusual branched Entner-Doudoroff (ED) pathway for sugar degradation that is promiscuous for glucose and galactose.<br>
As a part of the SulfoSYS project, this work focuses on the characterization of the following enzymes: glucose dehydrogenase (GDH-2, SSO3204), phosphoglycerate mutases (iPGAM, SSO0417 and dPGM, SSO2236), enolase (ENO, SSO0913), pyruvate kinase (PK, SSO0981), phosphoenolpyruvate synthetase (PEPS, SSO0831), pyruvate dikinase (PPDK, SSO2820) comprising cloning, expression and detailed analysis at different temperatures ranging from 50{\textdegree}C to 88{\textdegree}C. Furthermore, cell free extracts of S.solfataricus cells, grown at the temperatures 65{\textdegree}C, 70{\textdegree}C and 80{\textdegree}C, were prepared and analyzed for activity. In addition, temperature dependent stability of metabolic intermediates was detrmined. GDH-2, ENO, PK and PEPS, only annotated as putative enzymes in the S. Solfataricus genome, were confirmed as functional enzymes and characterized in detail.
S. solfataricus iPGAM was confirmed to exhibit activity in the anabolic direction in addition to the already known catabolic direction. The comparison of kinetic parameters suggests that GDH-2 might represent the major player in glucose catabolism via the branched ED pathway, while GDH-1 might have a role in the degradation of a variant of different sugars. The kinetic parameters of iPGAM, ENO, PK and PEPS show interesting trends regarding temperature change. Each enzyme behaves differently to changing temperature. It was shown that the metabolic intermediates PEP, NADH and NADPH are heat instable. Therefore a metabolic thermoadaptation strategy is essential for the organism.<br>
These findings, combined with the fact that the enzymes ENO and iPGAM have a different preference for the anabolic and the catabolic direction of the respective reaction, suggests a metabolic thermo adaptation by the control of flux of intermediates. The PEP producing step (ENO) is slowed down at higher temperatures while the PEP consuming step (PK) is accelerated at the same time, resulting in a relatively small amount of PEP at higher temperatures. Additionally, the PEP conversion to 2-PG by ENO is preferred at high temperatures. With changing catalytic parameters at different temperatures, this might allow the organism to keep pools of heat instable compounds small in order to avoid their decay.<br>
Furthermore, cell free extracts of S. solfataricus cells, grown at the temperatures 65{\textdegree}C, 70{\textdegree}C and 80{\textdegree}C, were analyzed for activity and in addition the temperature dependent stability of metabolic intermediates was investigated. Considering the general trend of substrate and co-substrate usage of the GDH reaction in S. solfataricus crude extracts it can be suggested that at least a third GDH isoenzyme or a specific galactose dehydrogenase is present in SSO.<br>
Kinetic data of activities in crude extracts, as well as of recombinant expressed enzymes were used to establish the first kinetic CCM model for an archaeal thermoacidophilic organism, which can be found at the following link:
http://jjj.biochem.sun.ac.za/sysmo/models/Sulfo-Sys/index.html
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  url = 	{https://duepublico2.uni-due.de/receive/duepublico_mods_00026846},
  file = 	{:https://duepublico2.uni-due.de/servlets/MCRFileNodeServlet/duepublico_derivate_00029011/Diss_Haferkamp.pdf:PDF},
  language = 	{en}
}