Cysteine thiolate

The final step in the /3-oxidation cycle is the cleavage of the /3-ketoacyI-CoA. This reaction, catalyzed by thiolase (also known as j8-ketothiolase), involves the attack of a cysteine thiolate from the enzyme on the /3-carbonyI carbon, followed by cleavage to give the etiolate of acetyl-CoA and an enzyme-thioester intermediate (Figure 24.17). Subsequent attack by the thiol group of a second CoA and departure of the cysteine thiolate yields a new (shorter) acyl-CoA. If the reaction in Figure 24.17 is read in reverse, it is easy to see that it is a Claisen condensation—an attack of the etiolate anion of acetyl-CoA on a thioester. Despite the formation of a second thioester, this reaction has a very favorable A).q, and it drives the three previous reactions of /3-oxidation. 

FIGURE 24.17 The mechanism of the thiolase reaction. Attack by an enzyme cysteine thiolate group at the /3-carbonyl carbon produces a tetrahedral intermediate, which decomposes with departure of acetyl-CoA, leaving an enzyme thioester intermediate. Attack by the thiol group of a second CoA yields a new (shortened) acyl-CoA. 

Metallothioneins (MT) are unique 7-kDa proteins containing 20 cysteine molecules bounded to seven zinc atoms, which form two clusters with bridging or terminal cysteine thiolates. A main function of MT is to serve as a source for the distribution of zinc in cells, and this function is connected with the MT redox activity, which is responsible for the regulation of binding and release of zinc. It has been shown that the release of zinc is stimulated by MT oxidation in the reaction with glutathione disulfide or other biological disulfides [334]. MT redox properties led to a suggestion that MT may possesses antioxidant activity. The mechanism of MT antioxidant activity is of a special interest in connection with the possible antioxidant effects of zinc. (Zinc can be substituted in MT by some other metals such as copper or cadmium, but Ca MT and Cu MT exhibit manly prooxidant activity.)... 

Based on the three-dimensional structure of CHS, we proposed that the initiation/elongation/cyclization cavity serves as a structural template that selectively stabilizes a particular folded conformation of the linear tetraketide, allowing the Claisen condensation to proceed from C6 to Cl of the reaction intermediate.14 In contrast, CTAL formation can occur either in solution or alternatively while sequestered in the enzyme active site. In either case, enolization of the C5 ketone followed by nucleophilic attack on the Cl ketone with either a hydroxyl group (in solution) or the cysteine thiolate (enzyme bound) as the leaving group results in CTAL. Similar lactones are commonly formed as by-products of in vitro reactions in other PKS systems.36 38... 

Figure 12.9 Repair of damaged DNA by sacrificial alkylation of one of the zinc cysteine thiolate ligands of the Ada DNA repair protein. (Reprinted with permission from Parkin, 2004. Copyright (2004) American Chemical Society.)...

In an article published independently at the same time, Stuehr and Ikeda-Saito89 used the purified bNOS and iNOS to reach the same conclusions. While the paper was under review, the authors mention that White and Marietta had reported earlier that the iron was a heme and was used as an oxidant. Using the same type of study, they found that the iron prophyrin and its CO derivative had the expected properties and proposed that the iron is penta-coordinated, with a cysteine thiolate as the fifth coordinate. A third publication confirmed the results when McMillan and coworkers90 used bNOS grown in human kidney cells. These workers obtained similar data for the light absorption of the enzyme and its CO spectrum. They also speculate on very similar sequences in the three types of purified enzyme that might be the porphyrin binding site. 

The serine hydrolases, threonine hydrolases, and cysteine hydrolases, the attacking nucleophile of which is a serine or threonine OH group or a cysteine thiolate group, respectively, and which form an intermediate covalent complex (i. e., the acylated enzyme). Here, an activated H20 molecule enters the catalytic cycle in the second step, i.e., hydrolysis of the covalent intermediate to regenerate the enzyme. 

Sharma and Reed, 1976)]. In proteins the coordination number 4 is most common, where the zinc ion is typically coordinated in tetrahedral or distorted tetrahedral fashion. The coordination polyhedron of structural zinc is dominated by cysteine thiolates, and the metal ion is typically sequestered from solvent by its molecular environment the coordination polyhedron of catalytic zinc is dominated by histidine ligands, and the metal ion is exposed to bulk solvent and typically binds a solvent molecule (Vallee and Auld, 1990). The inner-sphere coordination number of catalytic zinc may increase to 5 during the course of enzymatic turnover, and several five-coordinate zinc enzyme—substrate, enzyme product, and enzyme-inhibitor complexes have been studied by high-resolution X-ray crystallographic methods (reviewed by Matthews, 1988 Christianson and Lipscomb, 1989). The coordination polyhedron of zinc in five coordinate examples may tend toward either trigonal bipyramid or octahedral-minus-one geometry. 

Fig. 8.17. Mechanism of hydrolysis of phosphotyrosine residues by tyrosine phosphatases. Cleavage of phosphate from phosphotyrosine residues takes place by an in-line attack of a nucleophilic cysteine thiolate of the tyrosine phosphatase at the phosphate of the phosphotyrosine residue. The negative charge on the thiolate is stabilized by the positive charge of a conserved Arg residue. In the course of the reaction, an enzyme-Cys-phosphate intermediate is formed, which is hydrolytically cleaved to phosphate and enzyme-Cys-SH. The figure shows selected interactions. Other interactions in the active center involved in substrate binding and catalysis are not shown. According to Tainer and Russel, (1994). R substrate protein.

The blue color of these "type 1" copper proteins is much more intense than are the well known colors of the hydrated ion Cu(H20)42+ or of the more strongly absorbing Cu(NH3)42+. The blue color of these simple complexes arises from a transition of an electron from one d orbital to another within the copper atom. The absorption is somewhat more intense in copper peptide chelates of the type shown in Eq. 6-85. However, the -600 nm absorption bands of the blue proteins are an order of magnitude more intense, as is illustrated by the absorption spectrum of azurin (Fig. 23-8). The intense blue is thought to arise as a result of transfer of electronic charge from the cysteine thiolate to the Cu2+ ion.520 521... 

From EXAFS studies it has been concluded that there are at least two sulfur atoms at an average Mo—S distance of 2.41 A. Recent x-ray results of sulfite oxidase show that molybdenum is coordinated by two sulfur atoms of one MPT ligand and one sulfur from a cysteine thiolate. As deduced from the energy of the Raman active C=C and C—S stretching modes, there is considerable n delocalization in the 1,2-enedithiolate [139],... 

Type IIA The ligand donors come from ordinary protein residues, such as histidine imidazole, cysteine thiolate, and water (or hydroxide). 

There is a wider general interest in understanding the oxidation of cysteine thiolates in proteins since they are involved in redox-sensing reactions [99], Therefore, such oxidation reactions of thiols induced by Ru coordination may also play a more general role in the pharmacological activity of Ru-arene complexes by coupling Ru coordinative binding to redox processes both outside and inside cells. 

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