The energetic landscape of the allosteric regulatory mechanism of rabbit muscle pyruvate kinase (RMPK) was characterized by isothermal titration calorimetry (ITC). Four novel insights were uncovered. (1) ADP exhibits a dual property. Depending on the temperature, ADP can regulate RMPK activity by switching the enzyme to either the R or T state. (2) The assumption that ligand binding to RMPK is state-dependent is only correct for PEP but not Phe and ADP. (3) The effect of pH on the regulatory behavior of RMPK is partly due to the complex pattern of proton release or absorption linked to the multiple linked equilibria which govern the activity of the enzyme. (4) The R <--> T equilibrium is accompanied by a significant DeltaC(p), rendering RMPK most sensitive to temperature under physiological conditions. To rigorously test the validity of conclusions derived from the ITC data, in this study a fluorescence approach, albeit indirect, that tracks continuous structural perturbations was employed. Intrinsic Trp fluorescence of RMPK in the absence and presence of substrates phosphoenolpyruvate (PEP) and ADP, and the allosteric inhibitor Phe, was measured in the temperature range between 4 and 45 degrees C. For data analysis, the fluorescence data were complemented by ITC experiments to yield an extended data set allowing more complete characterization of the RMPK regulatory mechanism. Twenty-one thermodynamic parameters were derived to define the network of linked interactions involved in regulating the allosteric behavior of RMPK through global analysis of the ITC and fluorescent data sets. In this study, 27 independent curves with more than 1600 experimental points were globally analyzed. Consequently, the consensus results substantiate not only the conclusions derived from the ITC data but also structural information characterizing the transition between the active and inactive states of RMPK and the antagonism between ADP and Phe binding. The latter observation reveals a novel role for ADP in the allosteric regulation of RMPK.

• MeSH
• adenosindifosfát chemie   MeSH
• aktivace enzymů MeSH
• alosterická regulace MeSH
• chemické modely MeSH
• energetický metabolismus MeSH
• entropie MeSH
• fenylalanin chemie   MeSH
• fluorescenční spektrometrie * metody   MeSH
• fosfoenolpyruvát chemie   MeSH
• kosterní svaly enzymologie   MeSH
• králíci MeSH
• ligandy MeSH
• pyruvátkinasa antagonisté a inhibitory   chemie   metabolismus   fyziologie   MeSH
• terciární struktura proteinů MeSH
• tryptofan chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosindifosfát MeSH
• fenylalanin MeSH
• fosfoenolpyruvát MeSH
• ligandy MeSH
• pyruvátkinasa MeSH
• tryptofan MeSH

Rabbit muscle pyruvate kinase (RMPK) is an important allosteric enzyme of the glycolytic pathway catalyzing a transfer of the phosphate from phosphoenolpyruvate (PEP) to ADP. The energetic landscape of the allosteric regulatory mechanism of RMPK was characterized by isothermal titration calorimetry (ITC) in the temperature range from 4 to 45 degrees C. ITC data for RMPK binding to substrates PEP and ADP, for the allosteric inhibitor Phe, and for combination of ADP and Phe were globally analyzed. The thermodynamic parameters characterizing the linked-multiple-equilibrium system were extracted. Four novel insights were uncovered. (1) The binding preference of ADP for either the T or R state is temperature-dependent, namely, more favorable to the T and R states at high and low temperatures, respectively. This crossover of affinity toward R and T states implies that ADP plays a complex role in modulating the allosteric behavior of RMPK. Depending on the temperature, binding of ADP can regulate RMPK activity by favoring the enzyme to either the R or T state. (2) The binding of Phe is negatively coupled to that of ADP; i.e., Phe and ADP prefer not to bind to the same subunit of RMPK. (3) The release or absorption of protons linked to the various equilibria is specific to the particular reaction. As a consequence, pH will exert a complex effect on these linked equilibria, resulting in the proton being an allosteric regulatory ligand of RMPK. (4) The R <--> T equilibrium is accompanied by a significant DeltaC(p), rendering RMPK most sensitive to temperature under physiological conditions. During muscle activity, both pH and temperature fluctuations are known to happen; thus, results of this study are physiologically relevant.

• MeSH
• adenosindifosfát metabolismus   MeSH
• alosterická regulace MeSH
• chemické modely MeSH
• energetický metabolismus MeSH
• fenylalanin metabolismus   MeSH
• kalorimetrie * metody   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• kosterní svaly enzymologie   MeSH
• králíci MeSH
• ligandy MeSH
• pyruvátkinasa antagonisté a inhibitory   chemie   metabolismus   fyziologie   MeSH
• termodynamika * MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• adenosindifosfát MeSH
• fenylalanin MeSH
• ligandy MeSH
• pyruvátkinasa MeSH