Unisite ATP hydrolysis by soluble Rhodospirillum rubrum F1-ATPase is accelerated by Ca2+

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Soluble F1 (RF1) from Rhodospirillum rubrum is an ATPase enzyme.
• Divalent metal ions like Ca2+ and Mg2+ are crucial for RF1 activity.
• Previous studies indicated higher hydrolysis rates with Ca2+ at saturating ATP concentrations.

Purpose of the Study:

• To investigate the mechanisms behind RF1's higher catalytic activity with Ca2+ compared to Mg2+.
• To explore RF1's ATPase activity under unisite conditions (substoichiometric ATP concentrations).
• To determine the role of metal ions in ATP binding, hydrolysis, and product release.

Main Methods:

• Measuring ATPase activity of RF1 at substoichiometric [gamma-32P]ATP concentrations.
• Assessing the rate of [gamma-32P]ATP binding to RF1.
• Utilizing centrifugation-filtration through Sephadex columns to analyze enzyme-bound substrates and products.
• Comparing enzyme kinetics in the presence of Ca2+ versus Mg2+.

Main Results:

• Under unisite conditions, RF1 exhibited a 50-fold higher hydrolytic rate with Ca2+ than Mg2+.
• ATP binding rates were similar for both Ca2+ and Mg2+.
• Product release was significantly faster with Ca2+ than Mg2+.
• Cooperative mechanisms between catalytic sites were similarly affected by both cations.

Conclusions:

• The enhanced ATPase activity of RF1 with Ca2+ is primarily attributed to a decreased retention time of hydrolysis products.
• Ca2+ accelerates product release, leading to higher catalytic turnover rates.
• The study elucidates the specific role of Ca2+ in optimizing the catalytic cycle of RF1.