Consumption phosphate reactions

Endogenous NO is produced almost exclusively by L-arginine catabolism to L-citrul-line in a reaction catalyzed by a family of nitric oxide synthases (NOSs) [3]. In the first step, Arg is hydroxylated to an enzyme-bound intermediate "-hydroxy-1.-arginine (NHA), and 1 mol of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and O2 are consumed. In the second step, N H A is oxidized to citrulline and NO, with consumption of 0.5 mol of NADPH and 1 mol of 02 (Scheme 1.1). Oxygen activation in both steps is carried out by the enzyme-bound heme, which derives electrons from NADPH. Mammalian NOS consists of an N-terminal oxy-... 

Summary of anaerobic glycolysis. Reactions involving the production or consumption of ATP or NADH are indicated. The irreversible reactions of glycolysis are shown with thick arrows. DHAP = dihydroxyacetone phosphate. 

The oxygen consumption was monitored continuously with an oxygen electrode. The temperature was monitored simultaneously with a thermocouple immersed in the solution. At the start of the reaction 96 pmol NADH was added to 29.0 ml buffer containing 02. A nearly zero-order reaction was observed with the rate of 02 consumption of 6.87 pmol/min and the rate of temperature rise of 0.01171 K/ min. The heat capacity of the calorimeter and contents was 254.6 J/ K. What is AH for the above reaction NOTE The H+ is supplied by the phosphate buffer, which has a AH of dissociation of 5.4 kj mol-1. 

Procedures. Batch oxo experiments were performed in a 300cc autoclave. The autoclave and contents were flushed with CO and brought to reaction temperature at the beginning of a run. The vessel was then pressurized with the desired mixture of CO and hydrogen and the run commenced. Reaction was followed by monitoring hydrogen and CO consumption and by periodic sampling for GLC analysis on tricresyl-phosphate and Carbowax 1000 columns. 

A quantitative description of oxidative phosphorylation within the cellular environment can be obtained on the basis of nonequilibrium thermodynamics. For this we consider the simple and purely phenomenological scheme depicted in Fig. 1. The input potential X0 applied to the converter is the redox potential of the respiratory substrates produced in intermediary metabolism. The input flow J0 conjugate to the input force X0 is the net rate of oxygen consumption. The input potential is converted into the output potential Xp which is the phosphate potential Xp = -[AG hoS -I- RT ln(ATP/ADP P,)]. The output flow Jp conjugate to the output force Xp is the net rate of ATP synthesis. The ATP produced by the converter is used to drive the ATP-utilizing reactions in the cell which are summarized by the load conductance L,. Since the net flows of ATP are large in comparison to the total adenine nucleotide pool to be turned over in the cell, the flow Jp is essentially conservative. 

It now remained for us to apply the Coward protocol to our system and complete the synthesis of lipid I. Thus, phosphate 26 [Scheme 10], prepared by reductive cleavage of the phosphodiester protective groups of 9 (H2, Pd/C in MeOH, followed by pyridine, 91% yield), was converted to the corresponding phosphoroimidazolidate, whose formation was readily monitored via mass spectrometry. Excess carbonyldiimidazole was quenched via addition of methanol. The lipid phosphate salt was then added in portions via syringe until complete consumption of the phosphoroimidazolidate intermediate was observed. Mass spectrometry also allowed us to monitor the appearance of the desired lipid-linked diphosphate product. When the reaction was judged to be complete, the reaction solution was carefully concentrated and the crude product was treated with sodium hydroxide in aqueous dioxane in order to achieve global deprotection. The crude product was purified by reverse-phase... 

NADPH balances are often essential for metabolite balancing based estimations of the net fluxes in a metabolic network. However, NADPH consumption and generation are often found in bidirectional reactions that cannot be quantified by metabolite balancing approaches. The mannitol cycle (Fig. 9) is an example of a pathway that can affect the NADPH balance, but has no net conversion of any metabolites, except for cofactors. In the mannitol cycle, NADH and NADP+ are converted into NAD+ and NADPH, respectively, at the expense of ATP [52]. Because mannitol happens to be symmetrical, the activity of the mannitol cycle will cause scrambling of the carbon atoms of fructose 6-phosphate, and the activity of the cycle may therefore be identified using labeling analysis. The mannitol cycle has been reported to be present in several fungi [52]. 

Once citrulline is in the cytosol, argininosuccinic acid is formed by condensation of citrulline with aspartate. This is where the second nitrogen atom enters the cycle. Argininosuccinate synthetase, a homotetramer of a 46-kd polypeptide catalyzes the reversible reaction accompanied by hydrolysis of ATP to AMP and pyrophosphate. The subsequent hydrolysis of pyrophosphate shifts the equilibrium to the right and results in the consumption of two high-energy phosphate bonds. 

In biological systems, the most frequent mechanism of oxidation is the removal of hydrogen, and conversely, the addition of hydrogen is the common method of reduction. Nicotinamide-adenine dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP) are two coenzymes that assist in oxidation and reduction. These cofactors can shuttle between biochemical reactions so that one drives another, or their oxidation can be coupled to the formation of ATP. However, stepwise release or consumption of energy requires driving forces and losses at each step such that overall efficiency suffers. 

In contrast, the enzymes involved in the reduction of 3-PGA to triose phosphate together catalyse a freely reversible oxidation/reduction, the direction of which, in vivo, is largely determined by the levels of ATP and ADP, NADPH and NADP. In the light, with high levels of ATP and of NADPH the reactions proceed in the direction of triose phosphate driven by the production of 3-PGA and consumption of triose phosphate. In steady-state photosynthesis this provides for a coordination of the activity of parts of the cycle. Any component tending to increase the activity of PRK, for example, will cause the consumption of ATP and production of ADP. This in turn will slow the rate of 3-PGA reduction, leading to decreased synthesis of Rbu-5-P, bringing the cycle back into balance. 

The reaction begins with the phosphorylation of HCO3 to form carboxyphosphate, which then reacts with ammonium ion to form carbamic acid. Finally, a second molecule of ATP phosphorylates carbamic acid to carbamoyl phosphate. The structure and mechanism of the fascinating enzyme that catalyzes these reactions will be discussed in Chapter 25. The consumption of two molecules of ATP makes this synthesis of carbamoyl phosphate essentially irreversible. The mammalian enzyme requires H-acetyl-glutamate for activity, as will be discussed shortly. 

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