Common hydrogen bond interactions in diverse phosphoryl transfer active sites

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Phosphoryl transfer reactions are vital for cellular processes like energy metabolism and signaling.
• Previous studies focused on individual enzyme mechanisms, limiting comparative insights.
• Diverse phosphoryl transfer enzymes share conserved active site features.

Purpose of the Study:

• To develop a statistical approach for comparing active site configurations across diverse phosphoryl transfer enzymes.
• To identify conserved hydrogen bonding patterns that transcend enzyme families.
• To elucidate the mechanistic role of active site interactions in phosphoryl transfer.

Main Methods:

• Developed a top-down computational approach for statistical analysis of protein structures.
• Analyzed large datasets of enzyme structures from the Protein Data Bank.
• Performed ground-state quantum mechanical calculations on model compounds.
• Conducted experimental mutagenesis of phosphagen kinase (arginine kinase) to test predictions.

Main Results:

• Identified conserved hydrogen bonding patterns in active sites, particularly involving nucleotide O3β.
• Observed analogies in O3β interactions between phosphagen kinases and unrelated G proteins.
• Demonstrated that active site interactions modulate substrate phosphate charge and weaken the scissile bond via hyperconjugative effects.
• Mutating a key arginine residue in phosphagen kinase reduced catalytic rate (kcat) without significantly affecting substrate binding (KM).

Conclusions:

• Conserved hydrogen bonding patterns play a critical role in phosphoryl transfer across enzyme families.
• Active site interactions fine-tune substrate properties to facilitate catalysis.
• Database analysis combined with computational and experimental methods provides powerful insights into enzyme mechanisms.