Succinyl phosphate

The mechanism of succinyl-CoA synthetase is postulated to involve displacement of CoA by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active-site histidine (making a phosphohistidine intermediate) and release of succinate. The phosphoryl moiety is then transferred to GDP to form GTP (Figure 20.13). This sequence of steps preserves the energy of the thioester bond of succinyl-CoA in a series of high-energy intermediates that lead to a molecule of ATP ... 

D. Coenzyme A.—Succinyl phosphate (42) reacts rapidly and non-enzymatically with CoA in the pH range 3—8 to yield succinyl CoA (43). This reaction is dependent on the presence of a suitably situated free carboxy-group as such nucleophilic attack at carbon is not known with other acyl phosphates. Moreover, maleyl phosphate reacts rapidly with CoA while fumaryl phosphate fails to react under the same conditions. Hence the formation of a cyclic intermediate (44) from succinyl phosphate is... 

Examples of considerably more consequence are 1,3-diphosphoglyceric acid in the glycolytic pathway, and succinyl phosphate in the Krebs cycle. These compounds should not trouble us, since their reactivity is easily explained in terms of the above processes. 

In both cases, the mixed anhydride is used to synthesize ATP from ADP. Hydrolysis of the anhydride liberates more energy than the hydrolysis of ATP to ADP and, therefore, can be linked to the enzymic synthesis of ATP from ADP. This may be shown mechanistically as a hydroxyl group on ADP acting as nucleophile towards the mixed anhydride, and in each case a new phosphoric anhydride is formed. In the case of succinyl phosphate, it turns out that GDP rather than ADP attacks the acyl phosphate, and ATP production is a later step (see Section 15.3). These are enzymic reactions therefore, the reaction and the nature of the product are closely controlled. We need not concern ourselves why attack should be on the P=0 rather than on the C=0. 

The reaction is complex and involves an intermediate in which a phosphate is attached to a histidine residue of the succinate thiokinase enzyme. Probably CoA is first displaced by inorganic phosphate, forming succinyl phosphate. A nitrogen atom of a specific histidine residue then attacks phosphorus, displacing succinate and forming an A-phos-phoryl derivative. In the final step GDP attacks the phosphorus atom of that derivative forming GTP. The role of GTP in this reaction is played by ATP in some organisms. 

Oxoacyl-CoA transferase (see fig. 18.8) is involved in the transfer of a CoASH from succinyl-CoA to acetoacetate to produce succinate and aceto-acetyl-CoA. A cursory examination of this reaction suggests a simple transfer of the CoA moiety. However, it is soon realized that the loss of an oxygen by the acetoacetate and the gain of an oxygen by the succinyl group present a dilemma. Produce a rational mechanism that explains the preservation of the thio-ester energy and solves this dilemma. Hint Consider a succinyl phosphate intermediate. 

Many mechanisms are conceivable. One possibility is the approach of a phosphate on succinyl-CoA to produce succinyl phosphate (a mixed anhydride). 

This reaction is readily reversible (a near-equilibrium reaction) and has a AG of —0.7 kcal/mol (—2.9 kJ/mol). It proceeds with the formation of intermediates of the enzyme with succinyl phosphate and with phosphate (Pi), the latter being linked to histidyl residue of the enzyme (E) ... 

E-succinyl phosphate + CoASH E-succinyl phosphate E-phosphate -f succinate... 

Figure 17,13 Reaction mechanism of succinyl CoA synthetase. The reaction proceeds through a phosphorylated enzyme intermediate. (1) Orthophosphate displaces coenzyme A, which generates another energy-rich compound, succinyl phosphate. (2) A histidine residue removes the phosphoryl group with the concomitant generation of succinate and phosphohistidine. (3) The phosphohistidine residue then swings over to a bound nucleoside diphosphate, and (4) the phosphoryl group is transferred to form the nucleoside triphosphate.

The enzyme from E. coli utilizes ATP and ADP as substrates in place of the guanine nucleotides utilized by the mammalian enzymes. The mechanism of this reaction is reviewed in Volume X of this series (87). It is an interesting mechanism in that it involves an intermediate phosphoenzyme, with the phosphoryl group bonded to a histidine imidazole ring. The chemical reaction pathway consists of reactions (27a)-(27c), in which succinyl phosphate is an enzyme-bound intermediate. 

The importance of the phosphoenzyme in the mechanism of action of succinyl-CoA synthetase in reactions (27a)-(27c) is also unknown. The mechanisms of action of aminoacyl-tRNA synthetases and of acyl-CoA synthetases do not include covalent enzymic intermediates. The fact that succinyl-CoA synthetase involves succinyl phosphate as the activated substrate, whereas the others involve acyl adenylates, does not explain the difference. There is no chemical catalytic basis for the mechanisms of the formation of these intermediates to vary in this way. Moreover, acetate kinase produces acetyl phosphate without the intermediate formation of a phosphoenzyme, so that at least acetate kinase has the capacity to catalyze direct phosphorylation of a carboxylate group. 

The products suggest activation as a phosphoryl rather than as a nucleotidyl derivative. Both succinyl phosphate and thiophosphoryl coenzyme A have been suggested as intermediates. However, neither is included, at least as a freely dissociable intermediate, in current formulations of this reaction. An enzyme-bound phosphoryl histidine intermediate is thought to be involved, as is some sort of activated enzyme-CoA complex. Many aspects of the enzyme mechanism are still in doubt, but the sequence below is consistent with most available data. 

---