Pyrophosphate bond, hydrolysis

Ligases (syniheiases). Enzymes catalysing the joining together of two molecules coupled with the hydrolysis of a pyrophosphate bond in ADP or a similar triphosphate. They include some carboxylases and many enzymes known as synthetases. 

Isotope exchange experiments with purified F reveal a remarkable fact about the enzyme s catalytic mechanism on the enzyme surface, the reaction ADP + P, ATP + H20 is readily reversible—the free-energy change for ATP synthesis is close to zero When ATP is hydrolyzed by Fi in the presence of 180-labeled water, the Pj released contains an 180 atom. Careful measurement of the 180 content of P, formed in vitro by Fx-catalyzed hydrolysis of ATP reveals that the P, has not one, but three or four 180 atoms (Fig. 19-21). This indicates that the terminal pyrophosphate bond in ATP is cleaved and re-formed repeatedly before P, leaves the enzyme surface. With P, free to tumble in its binding site, each hydrolysis inserts 180 randomly at one of the... 

Because of coupling (see Chapter 7) there are relationships between the thermodynamic properties of reactions in some of the EC classes. All oxidoreductase reactions can be considered to be coupled reactions because each one can be divided into two, or in a some cases, three half reactions that do not share atoms but are connected by formal electrons. Transferase reactions can each be considered to result from the coupling of two oxidoreductase reactions or two hydrolase reactions. Fifteen examples are discussed in reference (6). Each of the coupled reactions contributes its A, G ° and A, A h to the coupled reaction. Hydrolase reactions and isomerase reactions are never coupled reactions. Some lyase reactions are coupled. Ligase reactions are all coupled by definition because they join together two reactions with the hydrolysis of a pyrophosphate bond in ATP or a similar triphosphate. A spectacular example of coupling is provided by EC 6.3.5.4 because there are seven reactants. This never happens in chemistry. 

Throughout the living world, energy is derived from the environment and used to support life by mechanisms that almost invariably end up depending, directly or indirectly, on the hydrolysis of the terminal pyrophosphate bond of ATP, giving ADP and inorganic phosphate (P,) ... 

Ribonuclease Hydrolysis of pyrophosphate bonds in ribonucleic acids Antiviral Spectrophotometry One unit produces soluble oligonucleotides equivalent to a change in absorbance at 260 nm of 1.0 in 30 min at pH 7.5 and 37°C in a 1.5 ml reaction volume... 

The third type of enzyme presumably involved in degradation of the polymer is phosphodiesterase. The snake venom enzyme has been used extensively to degrade the polymer for structural anedysis. The nuclear phosphodiesterase from liver will hydrolyze the pyrophosphate bonds of poly(ADP-ribose), NAD, NADH, and ADP-ribose 135). Futai has demonstrated that the hydrolysis of poly( ADP-ribose) proceeds from the AMP terminus 71) (refer to Fig. 4). 

Nicodnamide mononucleotide has been prepared from NAD by specific hydrolysis of the pyrophosphate bond using Zx ion as catalyst, 38 whilst NAD labelled with radioactive or stable isotopes,239 and the trifluoroacetyl analogue of NAD (COCF3 replacing CONHa) have been synthesized enzymically. Sih s laboratory has reported both enzymic and chemical routes to the NAD metabolite cyclic ADP-ribose (cADPR, 198). 

DPNasee. Besides the amino group of adenine, two other groups in the pyridine nucleotides are known to be sensitive to enzymatic attack. One is the nicotinamide-ribose bond and the other is the pyrophosphate bond. Nicotinamide is split from DPN by a group of widespread DPNases. The reaction catalyzed is a hydrolysis that results in the formation of free nicotinamide and a fragment abbreviated RPPRA. Some DPNases catalyze an exchange reaction in which nicotinamide from the medium enters the DPN molecule. In these systems nico-... 

Recent calculations which take into account the corrected value of 0.320 for the Ei value of the DPN-DPNH couplet show that the AFJ for the hydrolysis of the pyrophosphate bond in ATP is —8,640 calories (Anfinsen and Kielley, personal communication). If this value is correct, the AFo for the hydrolysis of thiolesters, acetyl-P and N-acetyl imidazole will be about —8,600, —11,200 and —14,000 calories respectively instead of the higher values which are reported in this paper. 

As an example we describe the methodology and data analysis to obtain the kinetic parameters for the PfdUTPase enzyme. dUTPase catalyzes the hydrolysis of a- -pyrophosphate bond of dUTP to yield dUMP and inorganic pyrophosphate (PPi). This... 

The values were AF ie = —8.5 kcal/mole, and AF 17 = -f-1 kcal/ mole. These calculations suggest that the free energy of hydrolysis of the methyl-sulfonium link is in the same order of magnitude as that of the pyrophosphate bonds of ATP, and justify inclusion of the sulfonium compounds in the list of energy-rich biological materials (Cantoni, 1960). 

Taking advantage of Lynen s spectrophotometric methods and the stability characteristics of thiolesters, it was readily shown that the incubation of phosphotransacetylase with acetyl-P and substrate levels of CoA led to the accumulation of a thiolester which was isolated and identified as acetyl-CoA.< > This represented the first net synthesis of acetyl-CoA in vitro and established once and for all the mechanism of the phosphotransacetylase catalyzed reaction. From equilibrium measurements of reaction (25), it was calculated that the standard free energy of hydrolysis of acetyl-CoA is about the same as that of the pyrophosphate bond of ATP (i.e. —8 kcal). The energy-rich nature of acetyl CoA was thus firmly established. 

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