Abstract
Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.
Highlights
Tryptophan fluorescence was investigateads a toolto Various suggestions have been made regarding thefunction study the noncatalytic nucleotide-binding sites of Es- of noncatalytic site-bound nucleotides, ranging from cofactors cherichia coli F,ATPase
ATP andADP, showed that nucleotide binding was macga-talytic site-bound and catalytic site-bound nucleotides and nesium-dependent, and showed that GTP and ITP did that it detect occupancy of noncatalytic sites by nucleotides bind to some extent, but AMP,GDP, and IDP did not
To determine whether the introduced mutations led to significant changes in the nucleotide binding properties of the noncatalytic sites, we measuredthe endogenous nucleotide content of the purified enzymes. aR365W mutant F, contained a total (ATP plus ADP) of 3.6 mol of nucleotide/mol of enzyme, pY354W mutant F, had 3.7,and wild-type F, had 3.8
Summary
Construction of aR365W and pY354W Mutations-The mutagenesis method was that of Vandeyar et al (1988) as described by Weber et al (1993a).For the uR365W mutation, the template was M13mp phage containing an SphI-Sal insert taken from plasmid pDP34 (Maggio et al, 1988). A introduces an MscI site while retaining proline at residue a366 The mutation was confirmed byDNA sequencing, transferred on an SfiIBstBI fragment from replicative form phage into plasmid pAW4 (Weber et al, 1993a1,and transformed into strain JP2 (AuncA)(Rao et al, 1988b).Transformants were screened on succinate plates and by PstI digestion to ascertain successful transfer of the mutation. Three candidate plasmids were transformed into strain JP17 (AuncD)(Lee et al, 1991).Strains containing each of these three plasmids had the same higher-than-wild-typegrowth yield in limiting glucose medium.One strain was designated SWM15. The plasmid from this strain (pSWM15) was shown byDNA sequencing to contain the pY354W mutation. Materials-lin-Benzo-ADP and lin-benzo-ATP were synthesized as described by Leonard etal. (1976, 1978).Concentration determinations for these analogswere based on an extinctioncoefficient of 9750 cm" a t 331 nm (Leonardet al., 1976)
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