Effect of phosphate and adenine nucleotides on the rate of labeling of functional groups at the catalytic site of F1-ATPase.

The effect of inorganic phosphate, ADP, ATP, and their analogues on the rate of labeling of F1-ATPase by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and phenylglyoxal have been investigated. Analysis of the kinetic data indicate that the labeled functional groups of the essential tyrosine and arginine residues respectively are both located at the catalytic site of F1. The active phenolic group of tyrosine is located closer to the bound inorganic phosphate or the gamma-phosphate group than the alpha- and beta-phosphate groups of the bound ATP at the catalytic site, whereas the guanidinium group of arginine is located closer to the alpha- and beta-phosphate groups of the bound ATP than to its gamma-phosphate group or the bound inorganic phosphate. The kinetically deduced dissociation constants are 1.3 mM and 210 microM for the inorganic phosphate and aDP respectively bound to this catalytic site. Labeling the essential tyrosine residue by NBD-Cl has been found to facilitate subsequent labeling of the essential arginine residue by phenylglyoxal.