H/D Exchange Characterization of Silent Coupling: Entropy-Enthalpy Compensation in Allostery

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein dynamics

Background:

• Allosteric systems involve effectors modulating substrate binding.
• Silent coupling occurs when enthalpy and entropy changes perfectly offset, masking affinity changes.
• Silent modulators are proposed as versatile drug leads.

Purpose of the Study:

• To investigate the temperature-dependent allosteric regulation of pyruvate kinase by alanine.
• To elucidate the molecular mechanism of silent coupling using hydrogen/deuterium exchange mass spectrometry (HDX-MS).
• To compare protein regions affected by silent coupling with those affected by allosteric inhibition.

Main Methods:

• Enzyme kinetics assays to measure substrate (phosphoenolpyruvate) affinity at different temperatures.
• Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) to probe protein conformational and dynamic changes.
• Allosteric regulation studies using alanine as an effector on rabbit muscle pyruvate kinase.

Main Results:

• Alanine reduced phosphoenolpyruvate affinity at 35°C and 10°C but was silent at intermediate temperatures.
• HDX-MS identified specific peptide regions undergoing changes during alanine-induced silent coupling.
• Affected regions in silent coupling partially overlapped with those identified for phenylalanine-induced allosteric inhibition.

Conclusions:

• Silent coupling in pyruvate kinase exhibits temperature-dependent behavior.
• HDX-MS is effective in mapping protein regions involved in silent allosteric regulation.
• Understanding silent coupling mechanisms can guide the rational design of allosteric drugs.