Thiol ester bond

Fig. 5.8. Model for catalytic role of E2 active-site asparagine. The side chain of the asparagine in the conserved HPN" motif (Figure 5.2) stabilizes the oxyanion that forms when the substrate s lysine attacks the E2/ubiquitin thiol ester bond. N79 is numbering for Ubcl (Figure 5.2).

There are at least three possibile ways in which the inhibitor can bind to the active site (1) formation of a sulfide bond to a cysteine residue, with elimination of hydrogen bromide [Eq. (10)], (2) formation of a thiol ester bond with a cysteine residue at the active site [Eq. (11)], and (3) formation of a salt between the carboxyl group of the inhibitor and some basic side chain of the enzyme [Eq. (12)]. To distinguish between these three possibilities, the mass numbers of the enzyme and enzyme-inhibitor complex were measured with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI). The mass number of the native AMDase was observed as 24766, which is in good agreement with the calculated value, 24734. An aqueous solution of a-bromo-phenylacetic acid was added to the enzyme solution, and the mass spectrum of the complex was measured after 10 minutes. The peak is observed at mass number 24967. If the inhibitor and the enzyme bind to form a sulfide with elimination of HBr, the mass number should be 24868, which is smaller by about one... 

Note that the enzyme catalyst enables the coupling of two chemically independent reactions the aldol condensation (with free energy change of about zero) to the very favourable hydrolysis of the CoA thiol ester bond which drives the overall reaction far towards product. Essentially, we have used an ATP s worth of energy to drive the reaction to completion. 

This is done in a two stage process involving first the addition of a third acetyl CoA by the second enzyme HMG-CoA synthase (2) via an aldol condensation (note the similarity to the formation of citrate in the TCA cycle, including driving the reaction by the hydrolysis of a thiol ester bond). 

The central role of thiol ester compounds in nutritional biochemistry is illustrated by considering some features of the mechanisms of the reactions catalyzed by citrate synthase and acctyl-CoA carboxylase. A brief background on the properties of the thiol ester bond is first presented. This tnaterial has been simplified and is intended only to clarify why the reactions employ thiol esters of carboxylic acids, rather than oxygen esters or the free, unesterified carboxylic acid. 

The PI anchor maintains adhesion of acetylcholinesterase (of the red blood cell) and of some proteoglycans (su I fated proteinsof the extracellular matrix) to the cell membrane Palmitic acid is bound via thiol-ester bonds toCys 322 and Cys 323 of rhodopsln (see the section on vitamin A), a 327-amino-add protein. The polypeptide chain of rhodopsin loops in and out of the membrane several times, leaving the possible function of the lipid as an anchor in question. Myristic add is bound to the catalytic subunit of the cAMP-dependent protein kinase, though this protein is cytosolic and soluble. 

G Protein is a class of proteins, occurring at the plasma membrane, that participates in transmitting signals from the extracellular fluid to various enzymes within the cell. Myristic acid is bound to G protein. Specifically, myristic acid is attached to the a subunit of G protein. This lipid is bound via an amide linkage to the N-terminal glycine. A geranylgeranyl group is attached via a thiol-ester bond to a cysteine residue on the y subunit of G protein (Casey and Seabra, 1996). 

In the first catabolic stage, digestion, food is broken down in the mouth, stomach, and small intestine by hydrolysis of ester, glycoside (acetal), and peptide (amide) bonds to jdeld primarily fatty acids, simple sugars, and amino acids. These smaller molecules are further degraded in the cytoplasm of cells to yield acetyl groups attached by a thiol ester bond (Section 21.9) to the large carrier molecule coenzyme A. The resultant compound, aceiyl coenzyme A acetyl CoA), is an intermediate in the breakdown of all main classes of food molecules. 

Like the serpins, AMG is a proteinase inhibitor. It is unlike the serpins in many aspects, however. First, it is a very large molecule, with a molecular mass of -725 kDa. As a result, only very small amounts diffuse out of the plasma space. Second, it acts as a substrate for proteases but does not block their active sites instead, it enfolds the still-active proteases to block access of proteins but not small substrates. Third, it inhibits many different classes of proteinases, mcluding those with serine, cysteine, and metal ions in their proteolytic sites. Fourth, it is structurally related to pregnancy zone protein and to the complement components C3, C4, and C5 rather than to the serpins. Like these proteins, it contains an intrachain thiol ester bond that is necessary for activity and the breaking of which results in a conformational change of the peptide chain. 

AMG is synthesized primarily by hepatic parenchymal cells. Catabolism is via two primary routes once the thiol ester bond is spht, AMG—regardless of whether complexed to a protease— is rapidly removed by a hepatic receptor that also acts to remove low-density lipoprotein. Desialylated... 

Although the two forms of AMG cannot be distinguished on routine electrophoresis, native AMG migrates slightly cathodal to AMG that has reacted with either proteases or nucleophilic substances that split the thiol ester bond. This characteristic can be used to determine the degree of inactivation that has occurred either in vitro or in vivo (e.g., in pancreatitis) monoclonal antibodies specific for the two forms can also be used in immunoassays for the same purpose. Using the latter assay, normal plasma contains 0.8% to 1.9% of AMG in the complexed form. ... 

C3 is the functional link between the classical and alternative pathways of activation and between these pathways and the membrane-attack complex (see Figure 20-7). It is also present in the highest concentration of all the complement components in plasma and acts as a magnification factor. Structurally and genetically, C3 is related to C4, C5, and AMG, aU of which contain an internal thiol ester bond that, when activated, can form complexes with membranes and other structures. 

C3 is synthesized as a propolypeptide that is cleaved post-synthetically to two disulfide-linked chains, a (molecular mass, HOkDa) and P (75kDa), During the process, an internal thiol ester bond is formed in the a-chain between adjacent glutamic acid and cysteine residues. C3 contains 3%... 

VIII. Utilization of Thiol Ester Bond Energy . 200... 

Thus far we have considered reactions in which there is a net synthesis of a thiol-ester bond. In addition to these, two different types of ester interchange reactions have been demonstrated by which one thiol ester can be used to synthesize another. One of these methods involves a transfer of the acyl group to other mercaptans, whereas the other involves a transfer of the thioalkyl group to various acids. 

In the foregoing discussions, we have considered those reactions which occur without a large change in free energy and emphasis has been placed on the fact that the reactions may lead to a net synthesis of thiol esters. It should be emphasized also that these reactions are for the most part freely reversible and that they may be regarded as reactions in which the energy of the thiol-ester bond is used for biosynthesis, viz., the synthesis of aldehydes, and keto acids. 

There is still another group of reactions, involving thiol-ester derivations of CoASH, which are highly exergonic in nature and are to be differentiated from those previously discussed in that they probably do not contribute significantly to the synthesis of thiol esters but instead represent reactions in w hich the enei of the thiol-ester bond is utilized more or less directly... 

Structure and bears a reactive -SH group that combines with acyl groups to form thiol esters. These are energy-rich compounds that can take part in reactions which would not be possible but for the favourable equilibrium established by cleavage of the thiol ester bond. 

Similarly the fatty acids must be activated by conversion to their CoA derivatives before they can be metabolized. Formation of the fatty acyl-CoA derivatives is catalysed by various/affy acid thiokinases (fatty acid CoA ligases) whose activity is linked with the breakdown of ATP to AMP and pyrophosphate, the liberated energy being used in the formation of the thiol ester bond ... 

An enzyme has been found in animal tissues and certain microorganisms which hydrolyzes the thiol ester bond of j8-hydroxy-/J-methylglutaryl CoA (mS). The most active source of this enzyme is chicken liver and this was the source used for purification. About a fourfold purification was achieved. 

Burton K (1955) The free energy change associated with the hydrolysis of the thiol ester bond of acetyl coenzyme A. Biochem J 59 44 6... 

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