Tryptophan-free Escherichia coli F1-ATPase.
We have engineered a mutant form of Escherichia coli F1-ATPase which is tryptophan-free and contains five mutations, namely delta W28L/alpha W513F/gamma W108Y/gamma W206Y/beta W107F. A strain carrying all five mutations grew normally by oxidative phosphorylation. Purified mutant F1-ATPase showed Vmax and Km both 65% higher than wild-type, resulting in kcat/Km the same as wild-type. The pH dependence of ATPase activity in mutant enzyme was very similar to that in wild-type. Catalytic-site nucleotide-binding characteristics were measured using the analog lin-benzo-ADP and sensitivity to inhibitors was tested using dicyclohexylcarbodiimide, azide and aurovertin. The mutant enzyme was very similar to wild-type in each of these characteristics. The fluorescence spectrum of mutant enzyme confirmed the absence of tryptophan. We have therefore established that it is possible to generate a tryptophan-free enzyme which retains normal catalytic function, oligomeric stability and in vivo assembly.
Wilke-Mounts
S
S
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Weber
J
J
Grell
E
E
Senior
A E
AE
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001313:Aurovertins / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001483:Base Sequence; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010085:Oxidative Phosphorylation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D001313:Aurovertins; D001386:Azides; D004024:Dicyclohexylcarbodiimide; C055520:linear-benzoadenosine diphosphate; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases