Metals, peptide bond hydrolysis

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-... 

Proteolytic cleavage (peptide bond hydrolysis) is another way that metal-mediated posttranslational modification of proteins may occur. Site-specific cleavage of polypeptides... 

Proteolytic cleavage (peptide bond hydrolysis) is another way that metal-mediated posttranslational modification of proteins may occur. Site-specific cleavage of polypeptides is essential for maturation of the protein and conversion of prohormones and cytokine precursor proteins to their biologically active forms, and proteolysis is also an important mechanism for regulating the protein activity through irreversible degradation. 

Four representative roles are conceivable for metals in metal-promoted peptide bond hydrolysis (Fig. 3) [31, 32]. First, metals act as a Lewis acid, and activate... 

In many metal ion-assisted hydrolytic peptide bond cleavage reactions, the metal ions, such as Pd, Pt, Cu, Mo, and Ni, are trapped by hydrolyzed products. Because free metal ions that are reactive for peptide bond hydrolysis hardly regenerate, examples for metal ion-catalyzed peptide bond cleavage are Umited. 

This zinc-dependent enzyme [EC 3.4.11.1], also referred to as cytosol aminopeptidase, leucyl aminopeptidase, and peptidase S, catalyzes the hydrolysis of a terminal peptide bond such that there is a release of an N-terminal amino acid, Xaa-Xbb-, in which Xaa is preferably a leucyl residue, but may be other aminoacyl residues including prolyl (although not arginyl or lysyl). Xbb may be prolyl. In addition, amino acid amides and methyl esters are also readily hydrolyzed, but the rates with arylamides are exceedingly slow. The enzyme is activated by heavy metal ions. 

Amino acids and their derivatives undergo a wide range of reactions, e.g. racemization, peptide bond formation, ester hydrolysis, aldol-type condensation, Schiff base formation and redox reactions, which are catalyzed by coordination to a metal centre. A number of reviews are available which cover some of these reactions.48,69,70... 

The ability of metal ions to catalyze the hydrolysis of peptide bonds has been known for 50 years, while the catalytic effect on the hydrolysis of amino acid esters was highlighted in the 1950s. As Hay and Morris point out in their review,76 the major problem with the kinetically labile systems is determining the nature of the reactive complex in solution. Such problems generally do not arise in the more inert systems and consequently reactions involving Co111 have been the more popular for study. 

Whilst metal-N(peptide) bond formation inhibits hydrolysis of the peptide bond, coordination to O(peptide) has the opposite effect. These differences in reactivity can be readily demonstrated and put to practical use with the inert Co111 complexes. One of the first examples was the reaction of [Co(trien)(H20)(OH)]2+ with peptides to give hydrolysis of the peptide bond at the N-terminal end. The proposed mechanism involving nucleophilic attack by hydroxide at the peptide carbon is shown in Scheme 7.110 Similar selective hydrolyses of N-terminal peptide bonds have since been demonstrated with other Co111 amine complexes and the reaction has been examined as a method for determining the N-terminal amino acid residue in peptides and proteins.1"112... 

Coordinated a-amino amides can be formed by the nucleophilic addition of amines to coordinated a-amino esters (see Chapter 7.4). This reaction forms the basis of attempts to use suitable metal coordination to promote peptide synthesis. Again, studies have been carried out using coordination of several metals and an interesting early example is amide formation on an amino acid imine complex of magnesium (equation 75).355 However, cobalt(III) complexes, because of their high kinetic stability, have received most serious investigation. These studies have been closely associated with those previously described for the hydrolysis of esters, amides and peptides. Whereas hydrolysis is observed when reactions are carried out in water, reactions in dimethyl-formamide or dimethyl sulfoxide result in peptide bond formation. These comparative results are illustrated in Scheme 91.356-358 The key intermediate (126) has also been reacted with dipeptide... 

To understand the role of metal ions in hydrolysis reactions, it is useful to first consider the background hydrolysis reactions. Table 6.1 lists the second-order rate constants for hydroxide-catalyzed hydrolysis of various substrates. The reactivity of methyl acetate (first entry in Table 6.1) [16] is comparable to those of other unactivated esters found in nature (e.g. acetyl choline and carboxyl esters in phospholipids). The reactivity of N-methylacetamide (second entry in Table 6.1) [17] is comparable to those of typical peptides (1.1 x 10 6 m-1 s 1) [18] and that of dimethyl phosphate (P-O bond... 

Fig. 2.18 Simplified scheme of the cycle/aUocation of magnesium in a green plant. Mg is involved - inter aha - in making peptide bonds, in tricarboxylate cycle, hydrolysis of molecules and binding of CO (ribulose-bisphosphatecarboxylase/oxidase or PEP carboxylase in plants which also employs Mg or Mtf+ in some plants (Kai et al. 2003)). Reaction steps in which Mg takes part as biocatalyst are marked by broken lines/arrows. Citrate and other intermediates of the tricarboxylate cycle, particularly malate, are employed by higher plants for extraction of essential metals, including Mg, Fe and Mn (thus the closed loop) from soil via and by means of the roots. This closed loop depicts a manner of autocatalysis. Amino acids which are required for protein biosynthesis are produced by reductive amination from tricarboxylate cycle intermediates and other 2-oxoadds which likewise eventually...

Cyclization is one of the earliest techniques applied to design peptidomimetics. Cyclic peptides are more stable to amide bond hydrolysis and allow less conformational flexibility consequently, the resulting analogs are anticipated to be more selective and less toxic. Methods for restricting conformations include peptide backbone cyclization, disulfide bond formation, side-chain cyclization, and metal ion chelation. 

V. Hydrolysis of Peptide Bonds by Catalysis with Metals and... 

Fig. 23. Hypothetical scheme (20) for hydrolysis of proteins by enzymes. Here glycylglycine is shown chelated (55o) through the metal M to an enzyme molecule (heavy line). If the latter contains a negatively charged group close to the peptide bond, hydrogen ions may be accumulated at the latter and so accelerate hydrolysis of this link.

Metal-bearing enzymes such as leucine amino peptidase catalyze the hydrolysis of N-terminal peptide bonds through a process involving chelation between the enzyme, the substrate, and the metal ion. CoHman reported the selective N-terminal hydrolysis of simple peptides by cis-hydroxyaquotriethylenetetramine cobalt (III)... 

There are several additional examples of zinc enzymes that contain an active site mononuclear zinc center and catalyze the hydrolysis of peptides bonds. These include carboxypeptidase B83 and neutral protease84 both of which contain a [(NHis)2(OGiu/AsP)Zn-OH2] coordination motif within the enzyme active site. Zinc peptidases that contain a [(NHis)3Zn-OH2] active site metal center include matrix metalloproteinases85,86 and other members of the metzincins superfamily.77,85... 

We have been involved in the synthesis of metal-based enzyme-like catalysts for hydrolysis of peptide bonds of proteins. We have been interested in hydrolysis as the target reaction since only one molecule is involved as the substrate, except for the water molecule. We selected peptide bonds as the targets since protein hydrolysis is important in the era of genomics. Proteomics and peptide bonds are quite stable with a half-life (26, 27) of spontaneous hydrolysis at (pH 7 at 25°C)... 

For designing enzyme-mimicking catalysts exploiting metal ions as catalytic centers, it is necessary to understand catalytic repertories (32, 33) of metal ions acting as Lewis acid catalysts in the hydrolysis of peptide bonds and related carboxyl derivatives (e.g., esters). Although esters are much easier to hydrolyze than peptides, important mechanistic information for catalysis in peptide hydrolysis can be obtained from that in ester hydrolysis. 

Amide nitrogen atoms of ordinary peptide bonds do not coordinate to metal ions since the nitrogen atom contains a partial positive charge due to resonance in the amide bond (F). In a p-lactam, the strained ring prohibits resonance and the nitrogen atom is basic enough to coordinate to a metal ion (G) leading to catalysis in amide hydrolysis (41, 42). 

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