Resonance energy transfer determination of the distance between the four cysteine-364 residues in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
Each of the four subunits of the Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase has one cysteine residue (Cys-364) that is protected against alkylation by MnATP and that is thought to be located at (or close to) the active site (M. Alvear, M. V. Encinas, S. Latshaw, R. G. Kemp, and E. Cardemil, 1992, Biochim. Biophys. Acta 1119, 35-38). To determine the distance relationships between these residues within this tetrameric enzyme, we have derivatized one of these reactive thiols with N-acetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine (AEDANS) and the others progressively with 4-[N-[(acetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (ANBD). In the doubly labeled protein nonradiative singlet-singlet energy transfer between AEDANS (donor) and ANBD (acceptor) was observed. The efficiency of the energy transfer is proportional to the number of occupied acceptor sites. From these data it has been determined that one of the acceptor sites is 33 A from the donor, and the remaining two sites are 44-46 A from the donor. Cross-linking experiments revealed that mainly cross-linked dimers were produced upon reaction of the enzyme with o-phthalaldehyde and dithiobissuccinimidylpropionate. We interpret these results as an indication that this tetrameric enzyme is most likely composed of an association of two dimers.
Alvear
M
M
Departamento de Ciencias Químicas, Facultad de Ingeniería y Administración, Universidad de la Frontera, Temuco, Chile.
Encinas
M V
MV
Herrera
L
L
Cardemil
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry*; D004735:Energy Transfer; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates; D010069:Oxadiazoles; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents
D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D010069:Oxadiazoles; D013439:Sulfhydryl Reagents; C015296:5-iodoacetamidofluorescein; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; C006421:1,5-I-AEDANS; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003545:Cysteine