Conformational flexibility and structure of creatine kinase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Structural biology

Background:

• Creatine kinase (CK) is crucial for cellular energy homeostasis.
• Understanding CK's structural flexibility is key to its function.
• Previous studies lacked detailed insights into dynamic conformational changes.

Purpose of the Study:

• To investigate the structural flexibility of creatine kinase.
• To characterize the active site environment using a hydrophobic fluorescent probe.
• To elucidate the effects of nucleotide and anion binding on enzyme conformation.

Main Methods:

• Utilized the covalent hydrophobic probe 2-[4'-(2"-iodoacetamido) phenyl] aminonaphthalene-6-sulfonic acid (IAANS).
• Measured fluorescence changes upon probe reaction with creatine kinase subunits.
• Assessed the impact of purine and pyrimidine nucleotides, and small anions on fluorescence.
• Employed fluorescence energy transfer (FRET) to determine active site distances.

Main Results:

• IAANS reacted differentially with creatine kinase subunits, indicating a hydrophobic cleft.
• Purine nucleotides enhanced IAANS fluorescence, while pyrimidine nucleotides quenched it.
• Small anions (e.g., chloride, nitrate) quenched both IAANS and tryptophan fluorescence.
• Anions non-competitively inhibited MgADP binding.
• FRET confirmed active sites are well-separated and move further apart upon nucleotide binding.

Conclusions:

• Creatine kinase exhibits significant structural flexibility.
• Nucleotide binding induces substantial conformational changes, altering active site accessibility.
• Anion binding near the active site influences enzyme conformation and nucleotide interactions.
• The study provides a detailed molecular mechanism for creatine kinase conformational dynamics.