Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Neuronal nitric oxide synthase (nNOS) plays a critical role in synthesizing nitric oxide (NO).
• Understanding the substrate binding kinetics of nNOS is essential for elucidating its regulatory mechanisms and function.
• Previous studies have indicated varying capacities of different substrates to support NO synthesis by nNOS.

Purpose of the Study:

• To characterize the binding kinetics of L-arginine and alternative substrates to nNOS.
• To elucidate the mechanism of substrate binding to nNOS.
• To correlate kinetic parameters with the substrate's capacity to support NO synthesis.

Main Methods:

• Utilized conventional and stopped-flow spectroscopy to monitor substrate binding.
• Employed imidazole displacement assay to track spectral changes in tetrahydrobiopterin-saturated nNOS.
• Analyzed kinetic data to derive rate constants and dissociation constants for substrate binding.

Main Results:

• Substrate binding to nNOS followed a monophasic, two-step reversible mechanism: Im-nNOS + S <=> Im-nNOS-S <=> nNOS'-S + Im.
• Kinetic parameters, including K(-)(1), k(2), k(-)(2), and apparent dissociation constants, were determined for L-arginine, homoarginine, N-methylarginine, and N-hydroxyarginine.
• Dissociation constants derived from kinetic data showed good agreement with equilibrium titration data.
• Significant differences in kinetic values were observed among the four substrates.

Conclusions:

• nNOS employs a rapid equilibrium followed by a slower isomerization step for substrate binding.
• The distinct kinetic profiles of L-arginine and its analogs suggest a basis for their varying efficiencies in supporting NO synthesis.
• These findings provide insights into the molecular mechanisms governing nNOS activity and substrate specificity.