Journal of Proteomics & Bioinformatics
Open Access
ISSN: 0974-276X
ISSN: 0974-276X
Sergei Khrapunov, Eric Chang, and Robert H Callender
Albert Einstein College of Medicine, USA
Posters & Accepted Abstracts: J Proteomics Bioinform
The thermodynamics of the apoenzyme, holoenzyme (LDH-NADH) and ternary (LDH-NADH-oxamate) complex of the glycolytic enzyme lactate dehydrogenase (LDH) from bsLDH (moderate thermophilic Bacillus stearothermophilus), porcine heart, phLDH (mesophilic Sus scrofa), and from mackerel icefish, cgLDH (psychrophilic Champsocephalus gunnari) have been investigated. A novel fluorescence assay was elaborated, which simultaneously monitors changes to the global protein structure, structural changes near the active site, and aggregation of the enzyme in response to increasing temperature. In our experiments the 2nd order of the monochromator grating was used to measure light scattering of the aggregated protein solution (the setup of 240 nm/470 nm excitation/ emission monochromators). Thus, three properties, light scattering, fluorescence resonance energy transfer (FRET), and NADH fluorescence could be measured simultaneously using respectively excitation at 240 nm, 280 nm, 340 nm and a fixed emission at 470 nm. The reverse changes of stability and affinity for oxamate were established for all orthologs. A reversible low-temperature (pre-denaturation) structural transition that precedes the high-temperature (denaturation) transition was found for the Michaelis complexes. This transition was found to coincide with a marked change in enzymatic activity for all LDHs. An observed lower substrate binding affinity for cgLDH compared to phLDH was accompanied by a higher contribution of entropy to �?G which reflects a higher functional plasticity of the psychrophilic cgLDH compared to the mesophilic phLDH. The comparative study of the apoenzyme and holoenzyme has shown that the basis for the pre-denaturation transition of the Michaelis complex is the flexibility of the global protein structure. The hypothesis is expressed that the multiple active and inactive along with intermediate sub-state conformations of the enzyme exist in equilibrium at the stage preceding irreversible thermal inactivation. This equilibrium is an essential selective factor for the adaptation of an enzyme to the environmental temperature. Recent Publications: 1. Khrapunov S, Chang E and Callender R H (2017) Thermodynamic and structural adaptation differences between the mesophilic and psychrophilic lactate dehydrogenases. Biochemistry 56:3587-3595.