Threonine peptides, hydrolysis

Data are based on spectral measurement of FAD in the solutions used for amino acid analyses. Corrections have been made for losses in serine and threonine upon hydrolysis by extrapolation of data at various times of hydrolysis to zero time and for the slow hydrolysis of some peptide bonds involving valine and isoleucine by using only those values obtained at later times of hydrolysis. 

Peptidases or proteases are enzymes that hydrolyse peptide bonds [9]. Proteolytic enzymes can be classified in five classes on basis of their catalytic mechanism aspartic, metallo-, cysteine, threonine and serine peptidases, whereby the latter three follow the same basic mechanism (Scheme 7.3) [10], Another classification of peptidases on the basis of statistically significant similarities in amino acid sequences was presented by Rawlings et al. (MEROPS database) [11], Serine proteases (SP) alone cover approximately one-third of all known proteases, and can accelerate the peptide hydrolysis very efficiently 10 fold) [6,11,12], SPs also hydro-... 

FIGURE 5.5 (a) The hydroxy amino acids serine and threonine are slowly destroyed during the course of protein hydrolysis for amino acid composition analysis. Extrapolation of the data back to time zero allows an accurate estimation of the amonnt of these amino acids originally present in the protein sample, (b) Peptide bonds involving hydrophobic amino acid residues snch as valine and isolencine resist hydrolysis by HCl. With time, these amino acids are released and their free concentrations approach a limiting value that can be approximated with reliability. 

Moser et al. (1968) (one of the co-authors was Clifford Matthews) reported a peptide synthesis using the HCN trimer aminomalonitrile, after pre-treatment in the form of a mild hydrolysis. IR spectra showed the typical nitrile bands (2,200 cm ) and imino-keto bands (1,650 cm ). Acid hydrolysis gave only glycine, while alkaline cleavage of the polymer afforded other amino acids, such as arginine, aspartic acid, threonine etc. The formation of the polymer could have occurred according to the scheme shown in Fig. 4.9. 

Whereas standard proteases use serine, cysteine, aspartate, or metals to cleave peptide bonds, the proteasome employs an unusual catalytic mechanism. N-terminal threonine residues are generated by self-removal of short peptide extensions from the active yS-subunits and act as nucleophiles during peptide-bond hydrolysis [23]. Given its unusual catalytic mechanism, it is not surprising that there are highly specific inhibitors of the proteasome. The fungal metabolite lactacystin and the bacterial product epoxomicin covalently modify the active-site threonines and in-... 

Proteasome jS-subunits are synthesized with N-terminal extensions and are inactive because a free N-terminal threonine is required for peptide-bond hydrolysis [130]. The precursor jS-subunits assemble with a-subunits to form half proteasomes com-... 

Peptides containing serine or threonine may undergo an N—>0 acyl shift upon exposure to strong acids (Scheme 40). 592,594 This reaction has been exploited in the structure elucidation of cyclosporin A, since the acid-catalyzed acyl shift with formation of an ester allowed its selective hydrolysis to the linear peptide for further stepwise degradation. 593 ... 

Alkaline hydrolysis with barium, sodium, or lithium hydroxides (0.2-4 M) at 110°C for 18-70 h126-291 requires special reaction vessels and handling. Reaction mixtures are neutralized after hydrolysis and barium ions have to be removed by precipitation as their carbonate or sulfate salts prior to analysis which leads to loss of hydrolysate. Correspondingly, peptide contents are difficult to perform by this procedure. Preferred conditions for alkaline hydrolysis are 4M LiOH at 145 °C for 4-8 h where >95% of tryptophan is recovered 291 An additional inconvenience of the alkaline hydrolysis procedure is the dilution effect in the neutralization step and thus the difficult application to the analyzer if micro-scale analysis is to be performed. The main advantage is the good recovery of tryptophan and of acid-labile amino acid derivatives such as tyrosine-0-sulfate1261 (Section 6.6) as well as partial recovery of phosphoamino acids, particularly of threonine- and tyrosine-O-phosphate (Section 6.5). 

Fig. 10. Postulated mechanism of hydrolysis of a peptide substrate by an aspartic proteinase. Stabilization of the tetrahedral intermediate depends heavily on hydrogen bonding interaction with serine and threonine residues (not shown). Reprinted with permission from James et al. (1992). Copyright 1992 American Chemical Society.

The amide bonds in peptides and proteins can be hydrolyzed in strong acid or base Treatment of a peptide or protein under either of these conditions yields a mixture of the constituent amino acids. Neither acid- nor base-catalyzed hydrolysis of a protein leads to ideal results because both tend to destroy some constituent ammo acids. Acid-catalyzed hydrolysis destroys tryptophan and cysteine, causes some loss of serine and threonine, and converts asparagine and glutamine to aspartic acid and glutamic acid, respectively. Base-catalyzed hydrolysis leads to destruction of serine, threonine, cysteine, and cystine and also results in racemization of the free amino acids. Because acid-catalyzed hydrolysis is less destructive, it is often the method of choice. The hydrolysis procedure consists of dissolving the protein sample in aqueous acid, usually 6 M HC1, and heating the solution in a sealed, evacuated vial at 100°C for 12 to 24 hours. 

Proteases are enzymes catalyzing the hydrolysis of peptide bonds. They form one of the largest enzyme families encoded by the human genome, with more than 500 active members. Based on the different catalytic mechanisms of substrate hydrolysis, these enzymes are divided into four major classes serine/threonine, cysteine, metallo, and aspartic proteases. In serine, cysteine, and threonine proteases, the nucleophile of the catalytic site is a side chain of an amino acid in the protease (covalent catalysis). In metallo and aspartic proteases, the nucleophile is a water molecule activated through the interaction with amino acid side chains in the catalytic site (non-covalent catalysis) (Gerhartz et al., 2002). 

The 20S proteasome is a protease complex comprising 28 subunits. It is a barrelshaped structure formed by the axial stacking of four rings made up of two outer a rings and two inner / rings arranged in / / order. Proteasomes are members of the N-terminal nucleophile- (Ntn-) hydrolase superfamily [19]. Their N-terminal threonine residues are exposed as the nucleophile in peptide bond hydrolysis [20, 21]. In eukaryotic cells, three of the fS-type subunits have N-terminal threonine residues, are active and have specificities determined largely by the nature of their... 

In general the thiol proteases catalyze the hydrolysis of a variety of peptide, ester, and amide bonds of synthetic substrates. Employing the general formula R —NH—CHR—CO—X, cleavage of the —CO—X— bond has been demonstrated when R represents the side chain of glycine, threonine, methionine, lysine, arginine, citrulline, leucine, and tyrosine. 

The assistance from a hydroxyl group in the hydrolysis of an amide bond either via the (largely reversible) hydroxyoxazolidine or the (largely irreversible) lactone mechanism leads to preferential cleavage of peptide bonds next to serine and threonine, on the one hand, and to lactones of... 

This was first foimd by Sanger et al. (1955) in a peptide from insulin and was observed with other peptides by Hirs et al. (1956) and Smyth et al. (1962). The reaction appears to occur when acidic buffers or dilute acids are employed for isolation of peptides. Conversion of the cyclic pyrrolidone carboxyl residue to a glutamyl residue is obtained on mild hydrolysis in dilute acids or alkalies. The cyclization reaction leads to difficulties when sequence methods are used which proceed from the amino-terminal end of a peptide. In addition, this reaction can occur when an internal glutamine residue becomes amino-terminal in the course of stepwise sequence analysis under acidic conditions, as in the Edman methods. An incorrect sequence for a peptide from ribonuclease was deduced as the result of cyclization of amino-terminal glutamine and acidic destruction of serine and threonine in the same peptide (Smyth et al., 1962). 

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