Mechanism for oxygen exchange in the chloroplast photophosphorylation system.

A method for analysis of positional isotope exchange (PIX) during ATP in equilibrium with HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the beta P-O gamma P bond. This cleavage yields Pi derived from the gamma-phosphoryl of ATP that contains all four of the gamma oxygens. Both PIX between the beta,gamma-bridge and beta-nonbridge positions and washout of the gamma-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17O at the beta-nonbridge positions and 18O at the beta,gamma-bridge and gamma-nonbridge positions. Application of this method to ATP in equilibrium with HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of gamma-nonbridge oxygens in the virtual absence of PIX. At 25 degrees C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s-1, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the beta-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 degrees C, this fraction corresponds to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. [Geeves, M. A., Webb, M. R., Midelfort, C. F., & Trentham, D. R. (1980) Biochemistry 19, 4748-4754] with subfragment 1.

Section: Discussioncontrasting

confidence: 87%

Section: Discussionmentioning

confidence: 89%

Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

Dale¹,

Hackney²

1987

“…The experimental results clearly show that a decrease in the NH4+ concentration during glutamine synthesis results in considerable oxygen exchange between glutamate and phosphate. Similar substrate modulations of oxygen exchange occur with E. coli succinyl-CoA synthetase , with soluble mitochondrial ATPase (Choate et al, 1979;, with membrane-bound ATPase (Russo et al, 1978), and in ATP synthesis by mitochondria and chloroplasts (Wimmer & Rose, 1977; Hackney & Boyer, 1978;Hackney et al, 1979). As has been shown with these systems, the most probable explanation of our results with glutamine synthetase is that the binding of NH4+ to a catalytic site of one subunit promotes catalytic events at another site.…”

Section: Discussionsupporting

confidence: 84%

Subunit interaction during catalysis: ammonium ion modulation of catalytic steps in the Escherichia coli glutamine synthetase reaction. Theoretical relationships for oxygen transfer catalyzed by Escherichia coli glutamine synthetase during net adenosine 5'-triphosphate cleavage

Gs¹,

Pd²

1980

A new approach for assessing if catalytic cooperativity may occur between subunits has been applied to Escherichia coli glutamine synthetase. The extent of oxygen exchange between bound [18O]glutamate and phosphate per molecule of glutamine formed was evaluated at various NH4+ concentrations. This allows calculation of the minimum number of reaction reversals in which bound glutamine is converted to bound glutamate prior to release of glutamine. At 1000 microM NH4+ no detectable reversals occurred, and only one glutamate oxygen appeared in product phosphate as required by the reaction mechanism. However, at 10 microM NH4+ over 15 reversals of bound glutamine formation occurred. Controls showed that under the experimental conditions free glutamine does not become significantly involved in exchange and, therefore, the reversal of the oxygen exchange steps appears to be limited to bound glutamine. In contrast to the effect seen with NH4+, adenosine 5'-triphosphate concentration appears to modulate the exchange of oxygen between glutamate and phosphate only slightly. These findings are interpreted as showing that NH4+ either promotes the dissociation of one of the reaction products or decreases the participation of bound products in the exchange. The NH4+ modulation of the oxygen exchange is consistent with binding of NH4+ at one catalytic site promoting catalytic events at an alternate catalytic site but does not eliminate all other explanations.

“…Another type of problem which can potentially be studied by the 31P (l70) method is the enzyme-catalyzed randomization of oxygen between the bridge and nonbridge positions, which often has significant mechanistic implications. This problem has been studied by using mass spectroscopic methods, as in the case of the mechanism of oxygen exchange between water and the -03 of ATP in the chloroplast phosphorylation system (Wimmer & Rose, 1977), and recently by the 31P NMR method based on the 180 isotope shift, as in the case of the mechanism of pyruvate kinase (Lowe & Sproat, 1978b).…”

Section: Discussionmentioning

confidence: 99%

Use of phosphorus-31 nuclear magnetic resonance to distinguish bridge and nonbridge oxygens of oxygen-17-enriched nucleoside triphosphates. Stereochemistry of acetate activation by acetyl coenzyme A synthetase

Tsai¹

1979

Adenosine 5'-(thiophosphate) AMPS) contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions. A general method was developed to distinguish between the "pro-R" and "pro-S" oxygens. When we converted the AMPS to the isomer A of adenosine 5'-(1-thiotriphosphate) (ATPalphaS), which is known to have S configuration at Palpha, the pro-R oxygen is incorporated into the bridge position, whereas the pro-S oxygen is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge oxygens based on the decrease in the peak intensity of 31P NMR signals caused by the directly bound 17O isotope. The method was used to elucidate the stereochemical course of acetate activation catalyzed by yeast acetyl coenzyme A (CoA) synthetase. The results indicate that yeast acetyl-CoA synthetase is specific for the isomer B of ATPalphaS and that the nucleophilic displacement proceeds with net inversion of configuration at Palpha of ATPalphaS (B), supporting the "in-line" mechanism.