Specificity of rabbit muscle phosphofructokinase for nucleotides as substrates or as allosteric effectors

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8-Bromoadenine nucleotides were tested as potential substrates and/or inhibitors of mitochondrial processes in intact or disrupted organelles, as substrates of various phosphotransferases, and as allosteric effectors in the reactions catalyzed by phosphofructokinase, isocitrate dehydrogenase, glutamate dehydrogenase, and fructose-1,6-bisphosphatase. 8-BrATP and 8-BrADP are not recognized by the translocase system located in the inner mitochondrial membrane and cannot be used as usbstrates in oxidative phosphorylation and related reactions catalyzed be beef heart submitochondrial membranes. This confirms the high specificity for adenine nucleotides of the mammalian systems involved in energy-yielding and energy-requiring reactions. However, 8-BrATP and 8-BrADP are able to substitute for the natural adenine nucleotides in reactions catalyzed by many phosphotransferases, although their capacity as phosphate donors and acceptors is generally much reduced. On the other hand, in almost all investigated cases, the 8-bromoadenine nucleotides have lost the capability of the natural adenine nucleotides to act as allosteric effectors, indicating that the structural requirements for allosteric activity are more stringent than those for catalytic activity.

Section: Resultsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 95%

mentioning

confidence: 99%

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Enzymic properties of 8-bromoadenine nucleotides

Lascu¹,

Kezdi²,

Goia³

et al. 1979

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“…1971a,b) was used to simulate the ATP enhancement of citrate binding. This analogue is not a substrate for phosphofructokinase, but it has been shown to bind to the enzyme (Wolfman et al, 1978;Pettigrew & Frieden, 1979) and inhibits its catalytic activity competitively with ATP (Barzu et al, 1977;Lad et al, 1977).…”

Section: Resultsmentioning

confidence: 99%

Interactions between citrate and nucleoside triphosphates in binding to phosphofructokinase

Ogawa¹,

Atkinson²

1985

Interrelationships between the binding by rabbit muscle phosphofructokinase of citrate, ATP, GTP, and adenyl-5'-yl imidodiphosphate (AMP-PNP) were investigated. To allow measurements at 25 degrees C, pyruvate kinase and phosphoenolpyruvate were included in the dialysis media to rephosphorylate ADP formed by the weak ATPase action of phosphofructokinase. Binding of citrate was enhanced by GTP nearly as much as by ATP, although GTP does not inhibit the catalytic activity of the enzyme. The results are consistent with the interpretation that binding of GTP, and, by analogy, ATP, at the catalytic site enhances the binding of citrate. AMP-PNP also enhanced citrate binding. Both ATP and GTP appear to bind at three sites per enzyme subunit, with the apparent third site binding relatively weakly. The estimated dissociation constants for the first two sites, about 33 microM for both for ATP compared with 3 and 280 microM for GTP, are consistent with kinetic results that imply lack of effective competition by GTP for the inhibitory site. When a compound binds at two or more sites on a macromolecule, the position and shape of the binding curve are sensitive to the geometric mean of the binding constants but quite insensitive to the magnitudes of the individual constants; thus, binding affinities cannot be estimated with confidence in such cases.

“…Phosphofructokinase has a broad specificity for NTP and high affinity for both substrates expressed by Km values below 0.1 mM (Bárzu et al, 1977). (b) Neither F6P nor FDP is hydrolyzed by the mitochondrial ATPase.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation and hydrolysis of 7-deazaadenine nucleotides by rat liver and beef heart mitochondria

Petrescu¹,

Lascu²,

Goia³

et al. 1982

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Tubercidin nucleotides [tubercidin 5'-mono-phosphate (TuMP), 5'-diphosphate (TuDP), and 5'-triphosphate (TuTP)] were tested as potential substrates for the mitochondrial phosphotransferases from rat liver and beef heart. TuDP is recognized by the mitochondrial translocase and phosphorylated by the respiratory chain enzymes in both mitochondria and submitochondrial particles from rat liver and beef heart; the low transport rate of the analogue into the matrix space of the intact organelles seems to be not a limiting step in the formation of TuTP. The phosphorylation of TuDP is significantly lower in beef heart mitochondria because of a higher specificity for ADP of the heart oxidative phosphorylation system. On the basis of the kinetic parameters of the partially purified liver mitochondrial adenylate kinase, one can conclude that the liver mitochondria are able to phosphorylate in vivo TuMP at a rate practically equal to the rate of AMP phosphorylation. The liver mitochondrial NDP kinase ensures a further fast phosphorylation of TuDP without the direct involvement of respiratory chain enzymes. In the case of heart mitochondria, two factors limit the rate of TuMP phosphorylation to TuTP: the lower acceptor activity of adenylate kinase with TuMP as compared with AMP and the different localization of heart NDP kinase situated on the inner face of the inner mitochondrial membrane. TuDP and TuTP preserve the ability of the natural nucleotides to interact with the "tight" nucleotide binding sites of isolated or membrane-bound F1. The low hydrolytic rate of TuTP with F1 may be related to the unusual flexibility of the glycosyl bond of tubercidin nucleotides in aqueous solution, with a high accessibility to syn conformation.