Cysteine enzymes

Enzyme modification. Polgar and Bender1,2 report use of this reagent for transforming the serine enzyme subtilisin-OH into the cysteine enzyme subtilisin-SH by the following process ... 

A Covalent Phosphoryl Cysteine Enzyme Intermediate in a Tyrosine Phosphatase 676... 

Disulfides. As shown in Figure 4, the and h-chains of insulin are connected by two disulfide bridges and there is an intrachain cycHc disulfide link on the -chain (see Insulin and other antidiabetic drugs). Vasopressin [9034-50-8] and oxytocin [50-56-6] also contain disulfide links (48). Oxidation of thiols to disulfides and reduction of the latter back to thiols are quite common and important in biological systems, eg, cysteine to cystine or reduced Hpoic acid to oxidized Hpoic acid. Many enzymes depend on free SH groups for activation—deactivation reactions. The oxidation—reduction of glutathione (Glu-Cys-Gly) depends on the sulfhydryl group from cysteine. 

Enzymatic Process. Chemically synthesized substrates can be converted to the corresponding amino acids by the catalytic action of an enzyme or the microbial cells as an enzyme source, t - Alanine production from L-aspartic acid, L-aspartic acid production from fumaric acid, L-cysteine production from DL-2-aminothiazoline-4-catboxyhc acid, D-phenylglycine (and D-/> -hydtoxyphenylglycine) production from DL-phenyUiydantoin (and DL-/)-hydroxyphenylhydantoin), and L-tryptophan production from indole and DL-serine have been in operation as commercial processes. Some of the other processes shown in Table 10 are at a technical level high enough to be useful for commercial production (24). Representative chemical reactions used ia the enzymatic process are shown ia Figure 6. 

The key enzyme in this sequence, isopenicillin N synthase (IPNS), has been purified from E. coli (59) and the recombinant enzyme shown to be a single polypeptide of 336 amino acids containing two cysteines, numbers 106 and 255 from the /V-teiminus, and probably a ferrous ion in a nonheme environment. The enzyme has been crystallized and studies undertaken to obtain suitably sized crystals for diffraction studies. 

Many enzymes need a certain ionic strength to maintain an optimum stabiHty and solubiHty, eg, bacterial a-amylases show optimal stabiHty in the presence of 1—2% NaCl. Some enzymes may need certain cations in low amounts for stabilization, eg, Ca " is known to stabilize subtiHsins and many bacterial a-amylases. Antioxidants (qv) such as sodium sulfite can stabilize cysteine-containing enzymes which, like papain, are often easily oxidized. 

Figure 1.9 Examples of functionally important intrinsic metal atoms in proteins, (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The iron atoms are bridged by a glutamic acid residue and a negatively charged oxygen atom called a p-oxo bridge. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules, (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains. During catalysis zinc binds an alcohol molecule in a suitable position for hydride transfer to the coenzyme moiety, a nicotinamide, [(a) Adapted from P. Nordlund et al., Nature 345 593-598, 1990.)...

Lysozyme from bacteriophage T4 is a 164 amino acid polypeptide chain that folds into two domains (Figure 17.3) There are no disulfide bridges the two cysteine residues in the amino acid sequence, Cys 54 and Cys 97, are far apart in the folded structure. The stability of both the wild-type and mutant proteins is expressed as the melting temperature, Tm, which is the temperature at which 50% of the enzyme is inactivated during reversible beat denat-uration. For the wild-type T4 lysozyme the Tm is 41.9 °C. 

In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. Cysteine residues in proteins, for example, react with one another to form disulfide species and also react with a number of reagents, including maleimides (typically A ethylmaleimide), as shown in Figure 4.11. Cysteines also react effectively... 

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. 

Cadmium is extremely toxic and accumulates in humans mainly in the kidneys and liver prolonged intake, even of very small amounts, leads to dysfunction of the kidneys. It acts by binding to the —SH group of cysteine residues in proteins and so inhibits SH enzymes. It can also inhibit the action of zinc enzymes by displacing the zinc. 

Step 1 of Figure 27.7 Claisen Condensation The first step in mevalonate biosynthesis is a Claisen condensation (Section 23.7) to yield acetoacetyl CoA, a reaction catalyzed by acetoacetyl-CoA acetyltransferase. An acetyl group is first bound to the enzyme by a nucleophilic acyl substitution reaction with a cysteine —SH group. Formation of an enolate ion from a second molecule of acetyl CoA, followed by Claisen condensation, then yields the product. 

The isomerization of isopentenyl diphosphate to dimethylally diphos phate is catalyzed by JPP isomerase and occurs through a carbocation pathway Protonation of the IPP double bond by a hydrogen-bonded cysteine residue ir the enzyme gives a tertiary carbocation intermediate, which is deprotonated b a glutamate residue as base to yield DMAPP. X-ray structural studies on the enzyme show that it holds the substrate in an unusually deep, well-protectec pocket to shield the highly reactive carbocation from reaction with solvent 01 other external substances. 

The retro-Claisen reaction occurs by initial nucleophilic addition of a cysteine -SH group on the enzyme to the keto group of the /3-ketoacyl CoA to yield an alkoxide ion intermediate. Cleavage of the C2-C3 bond then follows, with expulsion of an acetyl CoA enolate ion. Protonation of the enolate ion gives acetyl CoA, and the enzyme-bound acyl group undergoes nucleophilic acyl substitution by reaction with a molecule of coenzyme A. The chain-shortened acyl CoA that results then enters another round of tire /3-oxidation pathway for further degradation. 

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