Methanocaldococcus jannaschii adenylosuccinate synthetase: studies on temperature dependence of catalytic activity and structural stability.
Two important attributes of enzymes produced by thermophilic organisms are thermophilicity and structural stability. This manuscript discusses the characterization of these two aspects in adenylosuccinate synthetase from the thermophilic archaeon, Methanocaldococcus jannaschii. Adenylosuccinate synthetase catalyzes the formation of succinyl-AMP from IMP and aspartate with the simultaneous conversion of GTP to GDP. Temperature dependence of M. jannaschii AdSS (MjAdSS) catalysis exhibited a biphasic Arrhenius Plot with a transition at 40 degrees C. Pre-steady-state kinetics as a function of temperature indicated a change in rate determining step of the reaction across the inflection point. Slow release of products from the enzyme active site probably accounts for the thermophilicity of MjAdSS. Thermal unfolding of MjAdSS exhibited a T(m) of 85 degrees C, with the process being only partially reversible. Stability of MjAdSS assessed by equilibrium unfolding revealed the robustness of the secondary and tertiary structure of the enzyme which remained intact even at 8 M concentration of urea. Guanidinium chloride induced denaturation of MjAdSS permitted estimation of thermodynamic parameters. The unfolding profiles could be described as a composite of atleast two distinct transitions, with a stable intermediate in the unfolding pathway.
Mehrotra
Sonali
S
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore-560064, India.
Balaram
Hemalatha
H
D000265:Adenylosuccinate Synthase / Q000737:chemistry*; D004795:Enzyme Stability / Q000502:physiology; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D017016:Methanococcaceae / Q000201:enzymology*; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D014508:Urea / Q000737:chemistry
D014508:Urea; D000265:Adenylosuccinate Synthase; D019791:Guanidine