Thioester-Forming Steps

Metal complexes are known to insert CO into carbon-sulfur bonds [118], even catalytically [119], Stoichiometric precedents exist for the formation offhioesters from nickel-alkyls, CO, and thiols [120], For example, NiMe2(bipy) reacts with thiols to afford mefhylnickel(ll) fhiolates, which carbonylate to afford acetyl-nickel(ll) fhiolates. These acetylnickel(II) thiolates reductively eliminate fhioester in the presence of CO [121], More biologically relevant is the reactivity of nickel acyls toward fhiolates, which gives fhe thioester concomitant with reduction to Ni(0) (Eq. 12.10) [122]. Thiolates are known to reduce Ni(II) to Ni(0) under an atmosphere of CO [123]. 

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In a different approach, the reactivity of thiazolium salts derived acyl anion equivalents (biological active aldehyde ) toward sulfur electrophiles has been examined recently (329) and provides a model for the thioester-forming step catalyzed by the lipoic acid containing enzymes. The results suggest that the biological generation of thioesters of coenzyme A from a-keto acids occurs via the direct reductive acylation of enzyme-bound lipoic acid by the active aldehyde, as already shown on page 453. 

In the first step of the mechanism for this reaction, an SH group of a cysteine side chain at the active site of the enzyme reacts with glyceraldehyde-3-phosphate to form a tetrahedral intermediate. A side chain of the enzyme increases cysteine s nucleophilici-ty by acting as a general-base catalyst. The tetrahedral intermediate expels a hydride ion, transferring it to the 4-position of the pyridine ring of an NAD that is bonded to the enzyme at an adjacent site. NADH dissociates from the enzyme, and the enzyme binds a new NAD" ". Phosphate reacts with the thioester, forming the anhydride product... 

An example of an oxidation in which the oxygen atom is derived from the water molecule is the enzymic transformation of aldehydes to acids catalyzed by aldehyde dehydrogenases. In this case acyl thioesters formed in the oxidation step are converted to acids by hydrolysis (Eq. 12). 

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 ... 

Step 1 of Figure 29.3 Introduction of a Double Bond The /3-oxidation pathway begins when a fait)7 acid forms a thioester with coenzyme A to give a fatty acyl Co A. Two hydrogen atoms are then removed from C2 and C3 of the fatty acyl CoA by one of a family of acyl-CoA dehydrogenases to yield an a,/3-unsaturated acyl CoA. This kind of oxidation—the introduction of a conjugated double bond into a carbonyl compound—occurs frequently jn biochemical pathways and usually involves the coenzyme flavin adenine dinucleotide (FAD). Reduced FADH2 is the by-product. 

Step 4 of Figure 29.3 Chain Cleavage Acetyl CoA is split off from the chain in the final step of /3-oxidation, leaving an acyl CoA that is two carbon atoms shorter than the original. The reaction is catalyzed by /3-ketoacyl-CoA thiolase and is mechanistically the reverse of a Claisen condensation reaction (Section 23.7). In the forward direction, a Claisen condensation joins two esters together to form a /3-keto ester product. In the reverse direction, a retro-Claisen reaction splits a /3-keto ester (or /3-keto thioester) apart to form two esters (or two thioesters). 

Steps 6-8 of Figure 29.5 Reduction and Dehydration The ketone carbonyl group in acetoacetyl ACP is next reduced to the alcohol /S-hydroxybutyry] ACP by yS-keto thioester reductase and NADPH, a reducing coenzyme closely related to NADH. R Stereochemistry results at the newly formed chirality center in the /3-hydroxy thioester product. (Note that the systematic name of a butyryl group is biitanoyl.)... 

The process outlined above led to the provision of the necessary quantities of hydrogen for the reduction of CO2, CO, N03 and other oxidized starting materials, which were in turn converted to biomolecules in further reaction steps. The question as to whether contemporary living cells contain relicts, in the form of thioesters or thio compounds, which indicate the great importance of this class of substances, can clearly be answered positively. 

Because of the enzyme specificity in the hydration step, the new carbonyl group is introduced 3 to the original thioester carbonyl. The sequence includes two dehydrogenation reactions, and involves both FAD and NAD+ as coenzymes. The reduced forms of these coenzymes can be reoxidized by means of... 

Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis (AG ° = -36 kJ/mol). In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in either GTP or ATP, with a net AG ° of only -2.9 kJ/mol. Succinate is formed in the process ... 

The following experiments shed some light. Kinetic studies of sucdnyl-CoA-acetoacetate CoA transferase indicate a ping-pong mechanism. The enzyme alternates between two distinct forms, one of which has been shown to contain bound CoA.932-934 The E-CoA intermediate formed from enzyme plus acetoacetyl-CoA was reduced with 3H-containing sodium borohydride and the protein was completely hydrolyzed with HC1. Tritium-containing a-amino-8-hydroxyvaleric acid was isolated. Since thioesters (as well as oxygen esters) are cleaved in a two-step process... 

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