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ATP Binding to Noncatalytic Sites of Chloroplast Coupling Factor CF1

A. N. Malyan

Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia; fax: (0967) 790-532; E-mail: Malyan@issp.serpukhov.su

Received October 23, 2001

A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF1 was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF1 gamma-subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF1 with Mg2+ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant kd(ATP) . 10-3 min-1. Noncatalytic sites of CF1 were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF1 with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.

KEY WORDS:coupling factor CF1, noncatalytic sites, chloroplasts