Aspartate reaction mechanism

A number of iron-containing, ascorbate-requiring hydroxylases share a common reaction mechanism in which hydroxylation of the substrate is linked to decarboxylation of a-ketoglutarate (Figure 28-11). Many of these enzymes are involved in the modification of precursor proteins. Proline and lysine hydroxylases are required for the postsynthetic modification of procollagen to collagen, and prohne hydroxylase is also required in formation of osteocalcin and the Clq component of complement. Aspartate P-hydroxylase is required for the postsynthetic modification of the precursor of protein C, the vitamin K-dependent protease which hydrolyzes activated factor V in the blood clotting cascade. TrimethyUysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine. 

The carboxyl proteases are so called because they have two catalytically essential aspartate residues. They were formerly called acid proteases because most of them are active at low pH. The best-known member of the family is pepsin, which has the distinction of being the first enzyme to be named (in 1825, by T. Schwann). Other members are chymosin (rennin) cathepsin D Rhizopus-pepsin (from Rhizopus chinensis) penicillinopepsin (from Penicillium janthinel-lum) the enzyme from Endothia parasitica and renin, which is involved in the regulation of blood pressure. These constitute a homologous family, and all have an Mr of about 35 000. The aspartyl proteases have been thrown into prominence by the discovery of a retroviral subfamily, including one from HIV that is the target of therapy for AIDS. These are homodimers of subunits of about 100 residues.156,157 All the aspartyl proteases contain the two essential aspartyl residues. Their reaction mechanism is the most obscure of all the proteases, and there are no simple chemical models for guidance. 

Radkiewicz et al.184 explored the mechanism of aspartic acid racemization by means of the DFT(B3LYP)/SCRF calculations. The DFT/SCRF calculations provided quantitative rationalization of the rapid racemization observed at succinimide residues in proteins. The proposed reaction mechanism was supported by the computed increase of the acidity of the succinimide residue in aqueous solution compared to gas phase. 

Reactivity of Aspartic Acid Residues 6.3.3.1. Reaction Mechanisms... 

It is a common observation that a peptide bond formed by an aspartic acid residue is cleaved in dilute acids at a rate at least 100-fold faster than other peptide bonds. Three reaction mechanisms are now known to account for this specific reactivity [9] [90—95]. 

Fig. 6.27. Simplified representation of the reaction mechanisms by which aspartic acid residues can facilitate the hydrolytic cleavage of Asp-Xaa or Xaa-Asp peptide bonds. Pathway a begins as a nucleophilic attack of the C-flanking N-atom (i.e., the amide N-atom of the n+1 residue) at the carbonyl C-atom of the Asp side chain. Pathway b, which cleaves the Asp-Xaa bond, begins as a nucleophilic attack internal to the Asp residue. Pathway c, which cleaves the Xaa-Asp bond, is analogous to Pathway b, except that the attack is at the carbonyl C-atom of the N-flanking residue (i.e., the carbonyl of the n-1 residue).

The reaction mechanism of a-amylases is referred to as retaining, which means that the stereochemistry at the cleaved bond of the carbohydrate is retained. Hydrolysis of the glycosidic bond is mediated by an acid hydrolysis mechanism, which is in turn mediated by Aspl97 and Glu233 in pig pancreatic amylase. These interactions have been identified from X-ray crystallography. The aspartate residue has been shown to form a covalent bond with the Cl position of the substrate in X-ray structure of a complex formed by a structurally related glucosyltransferase. " The glutamate residue is located in vicinity to the chloride ion and acts as the acidic catalyst in the reaction. The catalytic site of a-amylases is located in a V-shaped depression on the surface of the enzyme. 

Aspartame is relatively unstable in solution, undergoing cyclisation by intramolecular self-aminolysis at pH values in excess of 2.0 [91]. This follows nucleophilic attack of the free base N-terminal amino group on the phenylalanine carboxyl group resulting in the formation of 3-methylenecarboxyl-6-benzyl-2, 5-diketopiperazine (DKP). The DKP further hydrolyses to L-aspartyl-L-phenyl-alanine and to L-phenylalanine-L-aspartate [92]. Grant and co-workers [93] have extensively investigated the solid-state stability of aspartame. At elevated temperatures, dehydration followed by loss of methanol and the resultant cyclisation to DKP were observed. The solid-state reaction mechanism was described as Prout-Tompkins kinetics (via nucleation control mechanism). 

Lipases belong to the subclass of a/P-hydrolases and their structure and reaction mechanism are well understood. All lipases possess an identical catalytic triad consisting of an aspartate or glutamate, a histidine, and a nucleophilic serine residue [67], The reaction mechanism of CALB is briefly discussed as a typical example of lipase catalysis (Scheme 7). 

The active site of serine proteases is characterized by a catalytic triad of serine, histidine, and aspartate. The mechanism of lipase action can be broken down into (i) adsorption of the lipase to the interface, responsible for the observed interfacial activation (ii) binding of substrate to enzyme (iii) chemical reaction and (iv) release of product(s). 

Belkai d, M., Penverne, B., Denis, M., and Herve, G. (1987). In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. 2. Reaction mechanism of aspartate transcarbamylase dissociated from carbamylphosphate synthetase by genetic alteration. Arch. Biochem,. Biophys., 254, 568-578. 

Hydrolytic activity of pepsin is maximal at pH 2-3, the stomach pH, and aspartate is an essential component of the active site. Pepsin is inactive at pH 7. Which enzymatic reaction mechanisms is most likely of major importance in the action of pepsin ... 

R) - and (S)-l-phenylethylamine to the diethyl esters of fumaric and maleic acid which are carried out by heating the pure compounds, without solvent, to 115-120 °C for three days (see Table 1). The reaction mixtures are then hydrolyzed and hydrogenated to give aspartic acids in high yields (85-87%) but very low optical purities (6.3-12.2%). A number of intermediates and by-products arc isolated, especially amides and imides of the dicarboxylic acids participating in the reaction. This may explain the low overall diastereoselectivity which can be calculated from the low optical rotation of the isolated aspartic acids. However, any discussion of the reaction mechanism remains difficult because the structures of the substrates and products of the actual addition step itself are not known with certainty. It is known that... 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

Serine carbohydrate esterases and transacylases. The commonest reaction mechanism is the standard serine esterase /protease mechanism, demonstrated paradigmally for chymotrypsin, involving an acyl-enzyme intermediate. The enzyme nucleophile is a serine hydroxyl, which is hydrogen bonded the imidazole of a histidine residue, whose other nitrogen is hydrogen bonded to a buried, but ionised, aspartate residue (Figure 6.28),... 

The protein is a 12-stranded anti-parallel p-barrel with amphipathic P-strands traversing the membrane (Fig. 4). The active-site catalytic residues are similar to a classical serine hydrolase triad except that in addition to the serine (Ser-144) and histidine (His-142), there is an asparagine (Asn-156) in place of the expected aspartic acid. Calcium at the active site is predicted to be involved in the reaction mechanism facilitating hydrolysis of the ester. 

The reaction mechanism of the SERCA- and PMCA-type Ca2+ pumps is essentially identical, involving the transient formation and hydrolysis of an acylphos-phate bond to an aspartate residue. For more information on the elementary steps of the reaction cycle the reader is referred to the reviews cited in the introduction and references therein. 

The decarboxylation of L-aspartic acid to L-alanine is catalysed by a pyridoxal-P-dependent j8-decarboxylase whose reaction mechanism is clearly different from that of the a-decarboxylases since the initial step probably involves C -H bond cleavage. The steric course at during the normal decarboxylation reaction has recently been shown [23b] to be inversion. In addition to this, however, the enzyme will also catalyse the decarboxylation of amino-malonic acid to glycine and Meister and coworkers [24,25] have shown that this process involves loss of the Si carboxyl group with overall retention at C . 

The reaction mechanism catalysed by sEH has been recently elucidated from experiments using heavy isotopes, protein, mass spectrometry, site-directed mutagenesis, and has been supported by the recent crystal structure determination at 2.8-A resolution (Fig. 31.28). This two-step reaction mechanism involves a catalytic nucleophile (aspartic acid 333) which can attack the polarized epoxide ring by two tyrosyl residues (tyrosines 381 and 465) leading to the ring opening and the formation of an acyl-enzyme intermediate. The second step corresponds to hydrolysis of this intermediate by a water molecule activated by a histidine 523-aspartic acid 495 pair." ... 

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