Cysteine, enzyme activation

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys— SH, cysteine thiol.) The— SH of the 4 -phosphopantetheine of one monomer is in close proximity to the— SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a "head-to-tail" arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

DNA sequence indicated that AMDase contains four cysteine residues located at 101, 148, 171 and 188 from amino terminal (Eig. 9). At least one of these four is estimated to play an essential role in the decarboxylation. The most effective way to determine which Cys is responsible to enzyme activity will be site-directed mutagenesis. To determine which amino acid should be introduced in place of active Cys, its role was estimated as illustrated in Eig. 13. One possibility is that... 

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... 

That myosin, a structural protein, also had enzyme activity as an ATPase, had been shown by Engelhardt and Ljubimova (1939-1941). ATP was now found to dissociate actomyosin producing a marked fall in viscosity the ATP was split to ADP and Pj. Contrasting properties of ATP in muscle systems were also observed. The rigor seen at postmortem occurred as ATP levels fell. The ATPase activity of myosin could be inhibited by mercurials (which block SH groups on cysteine) with ATPase blocked, ATP caused muscle fibers to relax (Weber and Portzehl, 1952). 

With isotopes it has been possible to show that all enzyme-catalyzed reactions are stereospecific. Before the availability of isotopes, there was no way of testing this generalization. Of course there are some apparent exceptions to prove the rule. Bently has listed a considerable number (2>, Table XIII, Chapter 6). The most interesting one to me seems to be luciferase, but that is an exception that isn t an exception. Thus, the enzyme luciferase acts on its substrate luciferin (2), in the presence of ATP and O2, to oxidize the luciferin to oxyluciferin (3). The reaction consists of an initial activation of the substrate by ATP to give luciferyl adenylate, after which the oxidation takes place. When the natural enantiomer (synthesized from D-cysteine) is activated and oxidized, light is emitted. The other enantiomer is also acted on by the enzyme, and is converted to the adenylate, but oxyluciferin is not formed, and there is no bioluminescence 37,38,38a)... 

In the previous studies using inhibitors and additives, it became clear that AMDase requires no cofactors, such as biotin, coenzyme A and ATP. It is also suggested that at least one of four cysteine residues plays an essential role in asymmetric decarboxylation. One possibility is that the free SH group of a cysteine residue activates the substrate in place of coenzyme A. Aiming at an approach to the mechanism of the new reaction, an active site-directed inhibitor was screened and its mode of interaction was studied. Also, site-directed mutagenesis of the gene coding the enzyme was performed in order to determine which Cys is located in the active site. 

Since the mode of inhibition is competitive and the Kj value is extraordinarily small compared to the value of the substrate (25 mM), it is strongly suggested that this inhibitor blocks the active site and prevents approach of the substrate to the catalytic site of the enzyme. It is assumed that a-bromo-phenylacetic acid interacts with a cysteine residue at the active site in some way. The high electron-withdrawing effect of the bromine atom would have an important role in the inhibition mechanism. Thus, the mode of binding of the inhibitor to the active site of the enzyme is presumed to resemble that of the substrate closely. Accordingly, disclosure of the way the inhibitor interacts with the enzyme would provide important information on how the enzyme activates the substrate. 

Aconitase was first determined to be an Fe-S protein in 1972 by Kennedy, Rauner and Gawron (23). Chemical analyses of inactive enzyme gave values of 2 Fe and 3 S /protein of 66,000 daltons. The observed molar relaxivity of water protons by this preparation of aconitase was 473 M s l (25). This value was an order of magnitude lower than measured in the earlier preparation of Villafranca and Mildvan (21) and much closer to that of Fe-S proteins (26). One mole of Fe + per mole of protein was taken up by the enzyme upon activation in the presence of cysteine and ascorbate, or lost upon inactivation in the presence of the iron chelator ferrozine (27). Gawron s group also demonstrated a correlation between loss of one Fe and loss of enzyme activity, as well as the protection afforded by citrate against both losses. However, the presence of an Fe-S cluster in aconitase remained for the moment a curiosity, in particular because of the unusual Fe/S= stoichiometries. The essential Fe that is correlated with activity continued to be interpreted in terms of the "ferrous-wheel" model. 

This enzyme contains one mole of NAD per mole of protein. The pyridine nucleotide can be released by the action of p-chloromer-curibenzoate, and the enzyme activity is lost by this treatment. The reverse process occurs418 when the inactivated enzyme is incubated with cysteine and NAD , and this procedure has been used to bind radioactive NAD to the enzyme.330 A requirement for NAD has been demonstrated for various similar enzymes from other sources.65,420... 

The enzyme activity is significantly inhibited by Tiron and 8-hydroxyquinoline, but not by a,a -dipyridyl and o-phenanthroline. The addition of thiol compounds such as cysteine, 2-mercaptoethanol and glutathione, and thiol inhibitors such as p-chloromercuribenzoate, N-ethylmaleimide and HgCl2 also markedly decreases the enzyme activity. The Michaelis constants of the enzyme are as follows 2-nitropropane (2.13 x 10-2M), nitroethane (2.43 x 10-2M), 3-nitro-2-pentanol (6.8 x 10 3M), 1-nitropropane (2.56 x 10-2 M) and oxygen (3.63 x 10 4M with 2-nitropropane)199. ... 

In their studies the enzyme activity decreased when reagents were added that complex with thiols hence it was concluded that cysteine was probably the key enzymatic nucleophile. The production of this enzyme could be induced by exposure of the bacteria to various phenylamide- or phenylurea-containing herbicides and fungicides. The enzyme was also capable of hydrolyzing a variety of other phenylamides and phenylureas, albeit at somewhat different rates. 

The mechanism of splicing is related to the chemistry of pyruvoyl enzyme activation (Eq. 14-41), succinimide formation from asparagine residues (Eq. 2-24), and protein carboxymethylation (Box 12-A). The intein always contains serine or cysteine in its N-terminal (l)-position and asparagine in its C-terminal position. The latter is always followed by cysteine, serine, or threonine in the N-terminal... 

Liver flukes also possess cathepsin C and LAP exopeptidases that are orthologous to the schistosome enzymes. These exopeptidases most likely complete the digestive process to yield free dipeptides and amino acids, respectively, from peptides generated by endoprote-olytic cysteine protease activity on host proteins. Both cathepsin C and LAP have been immunolocalized to gastrodermal cells (Carmona et al., 1994 Acosta et al., 1998 J.P. Dalton, unpublished data). 

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