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H S Penefsky

Publications and source records attributed to H S Penefsky.

At least 37 records · Page 2Linked to original sources
PubMed1981

The presence of two hydrolytic sites on beef heart mitochondrial adenosine triphosphatase.

The ribose-modified nucleotides 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP were used to probe the catalytic sites on soluble beef heart mitochondrial adenosine triphosphatase (F1). Both compounds were potent competitive inhibitors of ATP hydrolysis catalyzed by F1, Ki = 5.5 and 10 nM, respectively, and by submitochondrial particles, Ki (TNP-ATP) = 21 nM. Both compounds also were potent competitive inhibitors of ATP synthesis during oxidative phosphorylation (Ki = 1300 nM). Both analogs inhibited the 32Pi-ATP exchange reaction and the ATP-dependent reduction of NAD+ by succinate, catalyzed by submitochondrial particles. TNP-ATP and TNP-ADP were bound by F1. The presence of two binding sites on the enzyme for TNP-adenine nucleotides was determined by titrations of difference absorbance spectra, of the increase in fluorescence of the analog which occurred upon interaction with protein, and by titrations with the centrifuge column method using 32P-labeled TNP-adenine nucleotides. The first binding site bound the analogs with an affinity too high to be measured. The Kd for analog binding by the second site was 20 to 80 nM. In the presence of Mg2+, the 2 sites were filled with the TNP-ATP at a rate too rapid to be resolved by the procedure used. TNP-[gamma-32P]ATP was hydrolyzed by F1, Km = 0.2 microM, Vmax = 1.1 mol of 32Pi formed/mol of F1/s. It was shown, using the isotope trap technique as well as the inhibitor efrapeptin, that the 2 binding sites for TNP-ATP on F1 are hydrolytic sites.

Adenine Nucleotides↗
PubMed1981

Cooperatively between catalytic sites in the mechanism of action of beef heart mitochondrial adenosine triphosphatase.

Occupancy of only one of two hydrolytic sites on beef heart mitochondrial ATPase (F1) by the radioactive ATP analog, 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-[gamma-32P]-triphosphate (TNP-[gamma-32P]ATP) is associated with a low rate of hydrolysis of the substrate even under conditions otherwise favoring catalysis. Addition of excess nonradioactive TNP-ATP, in concentrations sufficient to fill catalytic Site 2 on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), accelerates the rate of hydrolysis of the radioactive substrate 15- to 20-fold. Since the excess nonradioactive substrate serves as an effective isotope trap, the involvement of medium TNP-[gamma-32P]-ATP as an intermediate is ruled out. These observations constitute direct evidence for catalytic cooperativity between active sites on F1. It is proposed that the use of high binding affinity substrates or substrate analogs, combined with the isotope trap technique, offers a new approach to the detection and study of catalytic site cooperativity in enzymes. The hydrolyzable nucleotides GTP, ITP, and ATP are excellent promoters of the hydrolysis of previously bound TNP-[gamma-32P]ATP whereas addition of nonhydrolyzable nucleotides such as TNP-ADP, ADP, and adenylyl imidodiphosphate result in a lower rate and extent of hydrolysis. AMP is without effect. Studies of the hydrolysis of [gamma-32P]ATP and TNP-[gamma-32P]ITP, under appropriate conditions, also provide evidence consistent with promoted catalysis. Based upon these findings, a model is presented for the mechanism of action of F1 in which site-site cooperativity reflects promoter-dependent hydrolysis of bound substrate.

Adenosine Triphosphatases↗
PubMed1981

Amino acid sequence of the protein inhibitor of mitochondrial adenosine triphosphatase.

The complete amino acid sequence of the mitochondrial ATPase inhibitor peptide was determined. The molecule contains 84 residues of which 40 are charged amino acids that occur in clusters along the chain. A section of the chain, located at the COOH-terminal end, contains several duplicated regions, the most prominent of which are pentapeptides. This section of the chain also contains all of the five histidines present in the molecule. Some of the physicochemical properties of the protein and an improved purification procedure are described.

Adenosine Triphosphatases↗
PubMed1980

The stereochemical course of phosphoric residue transfer catalyzed by beef heart mitochondrial ATPase.

The stereochemical course of phosphoric residue transfer has been determined for beef heart mitochondrial ATPase. When aden 5'-(3-thiotriphosphate), stereospecifically labeled with 18O in the gamma position, was hydrolyzed in [17O]water in the presence of the ATPase, the product inorganic [16O, 17O, 18O]thiophosphate was chiral. The configuration of the product showed that the hydrolysis had proceeded with inversion at the gamma-phosphorus atom. This result suggests that there is a direct, in-line transfer of the phosphoric residue between ADP and water and that there is no phosphoenzyme intermediate.

Adenosine Triphosphatases↗
PubMed1979

Mitochondrial ATPase.

Considerable progress has been made in recent years in our understanding of the phosphorylating apparatus in mitochondria, chloroplasts, and bacteria. It has become clear that the structure and the function of the ATP synthesizing apparatus in these widely divergent organisms is similar if not virtually identical. The subunit composition of F1, its molecular architecture, the location and function of substrate binding sites, as well as putative control sites, understanding of the component parts of the oligomycin-sensitive ATPase complex, and the role of these components in the function of the complex all are under active investigation in many laboratories. The developing information and the new insights provided have begun to permit experimental approaches, at the molecular level, to the mode of action of the ATPase in electron-transport-coupled ATP synthesis.

Adenosine Triphosphatases↗
PubMed1977

Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) exhibited a single binding site for Pi. The interaction with Pi was reversible, partially dependent on the presence of divalent metal ions, and characterized by a dissociation constant at pH 7.5 of 80 micronM. A variety of substances known to influence oxidative phosphorylation or the activity of the soluble ATPase (F1) also influenced Pi binding by the enzyme. Thus aurovertin, an inhibitor of oxidative phosphorylation, which was bound tightly by F1 and inhibited ATPase activity, enhanced Pi binding via a 4-fold increase in the affinity of the enzyme for Pi (KD = 20 micronM) but did not alter binding stoichiometry. Anions such as SO4(2-), SO3(2-), chromate, and 2,4-dinitrophenolate, which stimulated ATPase activity of F1, also enhanced Pi binding. Inhibitors of ATPase activity such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) inhibited Pi binding. The adenine nucleotides ADP, ATP, and the ATP analog adenylyl imidodiphosphate as well as the Pi analog arsenate, also inhibited Pi binding. The observations suggest that the Pi binding site was located in or near an adenine nucleotide binding site on the molecule.

Adenine Nucleotides↗
PubMed1975

Interaction of adenine nucleotides with multiple binding sites on beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) contained 2 mol of ADP and 1 mol of ATP/mol of enzyme, which resisted removal by Sephadex chromatography with dilute buffers or repeated precipitation with ammonium sulfate. The native enzyme also contained two apparently equivalent binding sites, which participated in readily reversible binding of adenyl-5'-ylimidodiphosphate (AMP-P(NH)P), with a Kd of 1.3 mum. The failure of AMP-P(NH)P to compete effectively with ADP for binding sites on F1 may be related to the failure of the analog to inhibit oxidative phosphorylation. Virtually complete removal of all adenine nucleotides from F1 occurred when the enzyme was chromatographed on columns of Sephadex equilibrated with 50% glycerol. No loss in ATPase activity was observed following removal of nucleotides from the enzyme, which was then capable of binding more than 4 mol of ADP and almost 5 mol of AMP-P(NH)P/mol of protein. Subsequent chromatography on columns of Sephadex equilibrated with dilute buffers containing Mg2+ removed only 1.5 mol of ADP and no AMP-P(NH)P from the enzyme. Reconstitution of F1 with ADP or with almost 5 mol of AMP-P(NH)P resulted in preparations that exhibited an undiminished capacity to restore oxidative phosphorylation in F1-deficient submitochondrial particles.

Adenine Nucleotides↗
PubMed1975

Physical and enzymatic properties of nucleotide-depleted beef heart mitochondrial adenosine triphosphatase.

Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1-deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1-deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.

Adenine Nucleotides↗