Characterization of the residues modified when F sub 1 - ATPases are inactivated by 7-chloro-4-nitrobenzofurazan and 5 prime -(p-(fluorosulfonyl)benzoyl)-1,N sup 6 -ethenoadenosine
Inactivation of the F{sub 1}-ATPases isolated from spinach chloroplasts (CF{sub 1}) and from the plasma membrane of the thermophilic bacterium, PS3 (TF{sub 1}) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) results in modification of Tyr-{beta}-328 and Tyr-{beta}-307, respectively. These residues are homologous to Tyr-{beta}-311 of the F{sub 1}-ATPase isolated from beef heart mitochondria, previously identified as the residue derivatized during inactivation of that enzyme with Nbf-Cl. Interestingly, an intramolecular migration of the Nbf- moiety from the tyrosine residue to a nearby lysine residue, observed when MF{sub 1} and TF{sub 1} which had been inactivated with Nbf-Cl are incubated at alkaline pH, was not observed when CF{sub 1} was treated in the same manner. CF{sub 1} differs from other ATPases in that it contains ADP, tightly bound at a single catalytic site. It is possible that this tightly bound ADP prevents migration of the Nbf moiety. The characteristics of inactivation of MF{sub 1} with the fluorosulfonyl benzoyl derivatives of adenosine (FSBA) and inosine (FSBI) have been described in the literature. Inactivation of MF{sub 1} with FSBA results in the mutually exclusive modification of Tyr-368 or His-427 in all three copies of the {beta} subunit. These residues comprise part of the noncatalytic nucleotide binding site. Inactivation of MF{sub 1} with FSBI results in modification of Tyr-{beta}-345 in a single catalytic site. The fluorosulfonyl benzoyl derivative of 1,N{sup 6}-ethenoadenosine (FSB{epsilon}A) has been prepared, and the characteristics and selectivity of modification of MF{sub 1} with this reagent are presented. FSB{epsilon}A binds reversibly to MF{sub 1} with an apparent dissociation constant of 250 {mu}M before covalent modification. The residue in MF{sub 1} that reacts with FSB{epsilon}A exhibits an apparent pK{sub a} of 8.9.
- Research Organization:
- California Univ., San Diego, CA (United States)
- OSTI ID:
- 5471907
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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ADENOSINE
BIOLOGICAL EFFECTS
ATP-ASE
ENZYME ACTIVITY
NITRO COMPOUNDS
AMINO ACID SEQUENCE
BACTERIA
CELL MEMBRANES
CHEMICAL COMPOSITION
CHLOROPLASTS
INHIBITION
METABOLISM
RESIDUES
SPINACH
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ACID ANHYDRASES
CELL CONSTITUENTS
ENZYMES
FOOD
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
MAGNOLIOPHYTA
MAGNOLIOPSIDA
MEMBRANES
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHOHYDROLASES
PLANTS
RIBOSIDES
VEGETABLES
560300* - Chemicals Metabolism & Toxicology
550501 - Metabolism- Tracer Techniques