Amino Group Of Cysteine Research Articles

Glucosamine-6-phosphate synthase from Escherichia coli: determination of the mechanism of inactivation by N3-fumaroyl-L-2,3-diaminopropionic derivatives

A mechanistic investigation of the inactivation of Escherichia coli glucosamine-6-phosphate synthase by N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionate (FMDP) was undertaken. On the basis of the known participation of the N-terminal cysteine residue in this process [Chmara et al. (1986) Biochim. Biophys. Acta 870, 357; Badet et al. (1988) Biochemistry 27, 2282], the model reactions between FMDP and L-cysteine and between FMDP and the synthetic decapeptide Cys-Gly-Ile-Val-Gly-Ala-Ile-Ala-Gln-Arg, corresponding to the amino-terminal protein sequence, were studied. The results allowed us to propose a pathway that is in perfect agreement with the biochemical results: enzyme inactivation arose from Michael addition of glutamine binding site cysteine-1 on the fumaroyl double bond at the beta-position of the ester group. Upon denaturation under slightly alkaline conditions, this adduct underwent cyclization to a transient succinimide adduct, which rearranged into the stable 2-substituted 1,4-thiazin-3-one-5-carboxylate involving participation of the cysteine amino group. The tryptic radiolabeled peptides purified from [3H]FMDP-treated enzyme and resistant to Edman degradation coeluted with the products resulting from the model reaction between the synthetic decapeptide and the inhibitor.

Prolonged antidiuretic action of vasopressin analogues in relation to their primary structure

The effectiveness of vasopressin analogues with prolonged antidiuretic action was related to their primary structure. Prolonged action was primarily due to the absence of the amino group of cysteine in position 1 of the peptide chain. The carba substitution of the disulphide bridge and introduction of basic homologous amino acids into position 8 contributed in differing degrees to the prolonged action of the analogues. [8-L-Norarginine]deamino vasopressin was the most potent of the analogues compared; its action was 10 times more prolonged than that of dDAVP.

The dehalogenation of iodouracil by cysteine. Intramolecular general-acid catalysis of cysteine addition to 5-iodouracil

The rate-determining step of the cysteine-catalyzed deiodination of 5-iodouracil is the formation of 5-iodo-6-cysteinyl-5,6-dihydrouracil. The rate of the reaction depends upon the concentration of un-ionized 5-iodouracil and the following ionic species of cysteine; −OOC(NH 3 +)CHCH 2S −. Unlike the reaction of 2-mercapto-ethanol with 5-iodouracil, the cysteine reaction is not subject to catalysis by imidazolium ion and tris(hydroxymethyl)aminomethane hydrochloride. When the rates of cysteine reacting with 5-iodouracil are measured in both H 2O and D 2O, a large kinetic isotope effect is observed ( k 2 H20 k 2 D20 = 4.10 ), thus implicating the protonated α amino group of cysteine as an intramolecular general acid catalyst for the reaction. These results and possible mechanisms for the actual dehalogenation of the intermediate 5-iodo-6-cysteinyl-5,6-dihydrouracil are discussed in terms of a possible mechanism for enzymatic halopyrimidine dehalogenation.

Cysteine-catalyzed Hydrogen Isotope Exchange at Position 5 of Uridylic Acid*

A new and potentially useful method for labeling nucleic acid was developed. Cysteine catalyzes hydrogen-deuterium exchange at position 5 of uridine 5′-phosphate under mild conditions. The pD dependence of the catalysis by cysteine, as well as that by 2-mercaptoethylamine showed a maximum at pD 8.9. Typical rates of the exchange at 37°C, determined by NMR spectroscopy were as follows: Reagent and its concentration/pD/the apparent rate constant (hr−1); cysteine, 0.5m/9.0/2.29×10−2; cysteine, 1.0m/9.0/6.05×10−1; 2-mercaptoethylamine, 0.5m/9.1/1.26x10–2; 2-mercapto-ethanol, 0.5m/9.5/1.51×10−3. N-Acetylcysteine, S-methylcysteine, or a mixture of the two agents was essentially ineffective in catalysis of the exchange. The amino group of cysteine is important in the catalysis since cysteine was much more effective than 2-mercaptoethanol as catalyst with 3-methyluridine at pD 10.0 where the SH-group of both compounds is largely dissociated. Based on these data, together with the known roles of a sulfur nucleophile and of amine in the bisulfite-catalyzed hydrogen isotope exchange of uridine (Y. Wataya and H. Hayatsu, Biochemistry, 11, 3583 (1972)), a reaction mechanism is proposed in which the mercapto anion adds across the 5, 6-double bond of the undissociated uracil moiety forming a 5, 6-dihydrouridine-6-mercapto compound and the NH2-group of cysteine abstracts the hydrogen at the 5-position intramolecularly. Studies on the effect of added amines and on the dependence of the exchange rate on the cysteine concentration suggested that protonated amines, either of added amine or of cysteine itself, accelerate the overall reaction. The acceleration is probably due to promotion of the addition of the SH to uridine. The implications of these findings in relation to the mechanism of the thymidylate synthetase reaction are discussed. This catalysis by cysteine appears to be the mildest method so far known for labeling uracil.

The incorporation of cysteine into an acceptor protein of human endometrium tissue

Studies indicate that components of a pathway for the incorporation of cysteine into preformed proteins are present in the 105 000 × g supernatant fraction of human endometrium. The incorporation required ATP and Mg 2+, but not tRNA, mRNA or ribosomes. The reaction is between the amino group of cysteine, and a specific protein or group of proteins with similar characteristics present in the supernatant fraction of the cell.

Glutathione

THE isolation by Hopkins of glutathione, the dipeptide of cysteine and glutaminic acid, was a definite advance in our knowledge of the processes of oxidation and reduction in living cells. The compound was found to exist in two forms, the reduced and the oxidised; the latter consists of two molecules of the former united by means of the sulphur atoms of their cysteine portions after the loss of two atoms of hydrogen. The oxidised form thus acts as a “hydrogen-acceptor”; in the reoxidation of this reduced form it is probable that active oxygen is set free for oxidation processes in the cell, oxygen also acting as the “hydrogen-acceptor”; whether the compound was present in the oxidised or reduced form seemed to depend largely on the hydrogen-ion concentration. It was suggested by Quastel, Stewart and Tunnicliffe that the dipeptide was formed by the linkage of the amino group of cysteine to that carboxyl group of glutaminic acid which is furthest away from its amino group; they based their conclusion on a study of the breakdown of the molecule in various directions.