Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-H2O oxygen exchange.

The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its gamma-PO3 oxygens with H2O before it dissociates from the enzyme. Pi, ASi, ADP, and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E X ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. Carbonyl cyanide m-chlorophenylhydrazone and NH4Cl (uncouplers of photophosphorylation) and phloridzin (an energy-transfer inhibitor) also lower the rate of ATP-H2O back-exchange; they too are found to act by decreasing the turnover of the ATP pool, not the extent of exchange per reacting ATP molecule. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form. The mode of NH4Cl inhibition of the ATP synthesis reaction, therefore, is not through a change in the partitioning of the E X ATP complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of ATPase of spinach coupling factor 1. Release of tightly bound ADP from the soluble enzyme.

Several seemingly unrelated procedures used to elicit the latent ATPase activity of soluble spinach coupling factor 1 can be correlated to the release of tightly-bound ADP from the uncoupled enzyme. This ADP release is further enhanced by the presence of medium nucleotides, especially substrate ATP, and may or may not involve release from the catalytic site itself. Similarly, the light/dithiothreitol activation of membrane-bound CF1 ATPase is reported to be accompanied by energy-dependent ADP dissociation. Further indication that ADP release is involved in the ATPase activation mechanism is the observation that a pyruvate kinase phosphoenolpyruvate trap for ADP released during light/dithiothreitol treatment greatly retards the decay of membrane-bound ATPase activity that occurs in the dark, presumably by preventing reassociation of ADP. The time course of CF1 reactivation by light, after light/dithiothreitol activation followed by dark decay, allows a distinction to be made between the apparently rate-limiting dithiol modification and the more rapid dissociation of tightly bound ADP.

Kinetic effects of chemical and physical uncoupling on the energy-transducing ATPase from spinach chloroplasts.

Ammonium chloride, an uncoupler of photophosphorylation which stimulates the membrane-bound chloroplast coupling factor ATPase when added after light/dithiothreitol activation, causes a decrease in the number of extra water oxygens incorporated into the phosphate formed during ATP hydrolysis. This observation is in contrast to the long-reported insensitivity of intermediate Pi:H2O oxygen exchange to uncoupler dinitrophenol in the mitochondrial F1 ATPase system. The effect of ammonium chloride on the CF1-catalyzed oxygen exchange reaction is consistent with ATPase activity stimulation caused by increased partitioning forward of the enzyme . products complex. In line with the oxygen exchange data, ammonium chloride causes an increase in the apparent Km of the enzyme for substrate ATP. The effect of ammonium chloride on the pattern of the intermediate Pi:H2O oxygen exchange is not a threshold phenomenon; the extent of exchange decreases in a continuous fashion, paralleling the stimulation of ATPase activity. The uncoupler CF3OPhzC(CN)2 also decreases the extent of oxygen exchange upon stimulating the membrane-bound ATPase, while phlorizin, an energy-transfer inhibitor, has essentially no effect on exchange although it inhibits the ATPase reaction. Similar to the effect of chemical uncoupling on the membrane-bound enzyme, physical removal of the coupling factor ATPase from the thylakoid membrane also results in an increase in forward partitioning of the enzyme . ADP . Pi complex. The modulation of oxygen exchange observed by altering the degree of coupling is similar to that which accompanies changing ATP concentration in the mitochondrial ATPase system [Russo, J. A., Lamos, C. M. and Mitchell, R. A. (1978) Biochemistry 17,473-480 and Choate, G. L., Hutton, R. L. and Boyer, P. D. (1979) J. Biol. Chem. 254, 286-290]. However, the uncoupler modulation is not readily correlated with the degree to which multiple catalytic sites are occupied by substrate.

Two types of kinetic regulation of the activated ATPase in the chloroplast photophosphorylation system.

The inhibition by light of chloroplast coupling factor ATPase is not due simply to competing photophosphorylation. This inhibition is only partially relieved by either an arsenate-pool trap for released phosphate, or a pyruvate kinase/phosphoenolpyruvate trap for ADP. Moreover, the amount of product return that does occur in the absence of trapping systems, ascertained by incorporation of 32Pi or [2-3H]ADP back into ATP during the hydrolysis reaction, is insufficient to account for the observed activity decrease. In intermediate pi:H2O oxygen exchange studies, the number of water oxygens incorporated into each molecule of Pi produced does not vary with light intensity during the ATPase assay. This indicates that the light-induced change in ATPase activity is not due to an alteration of rat constants involved in the forward and reverse partitioning of the E.ADP.Pi complex. In contrast, ammonium chloride, an uncoupler of photophosphorylation which stimulates membrane-bound coupling factor ATPase when added after light activation, causes a shift in the pattern of intermediate Pi:H2O oxygen exchange toward a lower number of water oxygens incorporated per Pi formed. The effect of NH4+ consistent with ATPase activity stimulation caused by enhanced partitioning forward of the E.products complex. These observations suggest the operation of two mechanisms of regulation of ATP ase activity during chloroplast de-energization. However, a direct effect of NH4+ on the coupling factor itself, independent of the membrane energization effect, cannot be ruled out by the present studies. Additional oxygen exchange experiments lead to the conclusion that the binding of ATP at a site catalyzing extensive ATP:H2O back exchange in the native chloroplast system ( Wimmer, M. J., and Rose, I. A. (1977) J. Biol. Chem. 252, 6769-6775) is different from the binding of ATP for net hydrolysis in the system activated for ATPase.

A suggestion for naming faces of ring compounds.

Discussion of the topology of interaction of ring compounds with macromolecules and receptors requires a system for naming the faces of the cyclic compound. An alpha/beta-face nomenclature is suggested that is based on the clockwise/counterclockwise direction of ascending numbering in the ring with the lowest numbered unshared ring atom. This system is applicable to a very broad range of compounds: sugars, cyclic bases, steroids, cyclitols, porphyrins, etc., and molecules with a single ring, fused rings, encompassing rings, and rings formed by head-to-tail polymerization.

Mechanism for oxygen exchange in the chloroplast photophosphorylation system.

The oxygen exchange that occurs between water and the gamma-PO3 of ATP in light-activated chloroplast lamellae was found to proceed with close to full equilibration of the oxygens before ATP returned to the medium. This is in contrast to the entry of approximately one water oxygen when ATP is synthesized from ADP and P1 in the same system. In the latter case, the limitation is kinetic, however, not steric, as shown by the presence of some molecules containing more than one water-derived oxygen in the gamma-PO3. The different extents of exchange can be explained by a relatively faster rate of dissociation of ATP from the chloroplast coupling factor during synthesis from ADP and P1 relative to its dissociation in the absence of net phosphorylation. To determine the mechanism of gamma-PO3:H2O exchange, its rate was compared with the rate of reversible cleavage of ATP as detected by betagamma bridge to beta nonbridge 18O scrambling in [Pbeta-18O-Pgamma]ATP (Midelfort, C. F., and Rose, I. A. (1976) J. Biol. Chem. 251, 5881-5887). The scrambling reaction, which depends on cleavage of the PbetaO--Pgamma bond, was found to occur in nearly the same fraction of ATP molecules that experienced gamma-PO3:H2O exchange in the same incubation, suggesting that the latter is due to multiple cycles of reversible ATP hydrolysis on the chloroplast coupling factor, i.e. [ATP-H2O in equilibrium ADP-Pi].

Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.

Biphasic inactivation of procine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate.

Temperature studies have indicated that from 0 to 37 degrees, the time-dependent inactivation of mitochondrial malate dehydrogenase from porcine heart by pyridoxal 5-phosphate (pyridoxal-5-P) is biphasic. The initial phase of the inactivation is reversible but can be made irreversible by reduction with sodium borohydride. The reduced pryidoxal-5-P-enzyme adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325. The irreversible second phase of the inactivation is accompanied by the appearance of a new 325-nm absorbance maximum, in the absence of reduction, and a fluorescence emission centered about 390 to 400 nm when excited at 325. The evidence presented suggests the formation of a Schiff base between pyridoxal-5-P and a nucleophilic residue, most likely lysine, of malate dehydrogenase during the first phase of inactivation. An X-azolidine-like structure, a further derivative of the Schiff base, possessing spectral properties consistent with the reported data, may be formed during the second phase; this presumably involves a second nucleophilic residue of the enzyme, implicating the action of pyridoxal-5-P as a bifunctional reagent in this instance. The presence of the coenzyme, NADH, protects the enzyme from inactivation, suggesting that pyridoxal-5-P interacts at or near the malate dehydrogenase active center. Simultaneous binding studies using pyridoxal-5-P with known malate dehydrogenase competitive inhibitors AMP, ADP, and nicotinamide indicate that the pyridoxal-5-P modification occurs in the general area of the ADP portion of the coenzyme binging site. Furthermore, the presence of nicotinamide enhances pyridoxal-5-P binding to and inactivation of malate dehydrogenase.