Characterization and function of catalytic subunit a of H/sup +/-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae
Subunit a (M/sub r/ 89,000) from vacuolar membrane H/sup +/-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae was found to bind 8-azido(..cap alpha..-/sup 32/P)adenosine triphosphate. Labeling by this photosensitive ATP derivative was saturable with an apparent dissociation constant of 10/sup -6/ to 10/sup -5/ M and decreased in the presence of ATP and ADP. The enzyme was inactivated by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), with about 1 ..mu..M causing half-maximal inactivation in the neutral pH range. This inactivation was prevented by the presence of ATP, ADP, or adenosyl-5'-yl imidodiphosphate (AMP-PNP). The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol. Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme. When the enzyme was inactivated with (/sup 14/C)NBD-Cl, subunit a was specifically labeled, and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP. From these results, it was concluded that subunit a of yeast vacuolar H/sup +/-ATPase has a catalytic site that contains a single, essential tyrosine residue. The kinetics of single site hydrolysis of (..gamma..-/sup 32/P)ATP indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and P/sub i/ at the NBD-Cl-sensitive catalytic site.
- Research Organization:
- Univ. of Tokyo (Japan)
- OSTI ID:
- 7075858
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:1; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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560300 -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ACID ANHYDRASES
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
CELL CONSTITUENTS
CELL MEMBRANES
DAYS LIVING RADIOISOTOPES
ENZYMES
FUNGI
HYDROLASES
ISOTOPES
KINETICS
LIGHT NUCLEI
MEMBRANES
MICROORGANISMS
NITRO COMPOUNDS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PH VALUE
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PLANTS
RADIOISOTOPES
REACTION KINETICS
SACCHAROMYCES
SACCHAROMYCES CEREVISIAE
YEASTS