NUCLEOTIDE BINDING TO THE ISOLATED BETA-SUBUNIT OF THE CHLOROPLAST ATP SYNTHASE

The beta-subunit isolated from the chloroplast ATP synthase F1 (CF1) has a single dissociable nucleotide binding site, consistent with the proposed function of this subunit in nucleotide binding and catalysis. The beta-subunit bound the nucleotide analogs trinitrophenyl-ATP (TNP-ATP) or trinitrophenyl-ADP (TNP-ADP) with nearly equal affinities (K(d) = 1-2-mu-M) but did not bind trinitrophenyl-AMP. Both ATP and ADP effectively competed with TNP-ATP for binding. Other nucleoside triphosphates were also able to compete with TNP-ATP for binding to beta; their order of effectiveness (ATP > GTP, ITP > CTP) mimicked the normal substrate specificity of CF1. The single nucleotide binding site on the isolated beta-subunit very closely resembles the low affinity catalytic site (site 3) of CF1 (Bruist, M. F., and Hammes, G. G. (1981) Biochemistry 20, 6298-6305), suggesting that tight nucleotide binding by other sites on the enzyme involves other CF1 subunits in addition to the beta-subunit. The results are inconsistent with an earlier report (Frasch, W. D., Green, J., Caguil, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069), which suggested more than one nucleotide binding site per beta-subunit. Binding of nucleotides to the isolated beta-subunit was eliminated by a brief heat treatment (40-degrees-C for 10 min) of the protein. A small change in the circular dichroism spectrum of beta-accompanied the heat treatment indicating that a localized (rather than global) change in the folding of beta, involving at least part of the nucleotide binding domain, had occurred. Also accompanying the loss of nucleotide binding was a loss of the reconstitutive capacity of the beta-subunit. ATP protected against the effects of the heattreatment.