Aspartate residues

FIGURE 16.27 A mechanism for the aspartic proteases. In the first step, two concerted proton transfers facilitate nucleophilic attack of water on the substrate carbonyl carbon. In the third step, one aspartate residue (Asp" " in pepsin) accepts a proton from one of the hydroxyl groups of the amine dihydrate, and the other aspartate (Asp" ) donates a proton to the nitrogen of the departing amine. 

The HIV-l protease is a remarkable viral imitation of mammalian aspartic proteases It is a dimer of identical subunits that mimics the two-lobed monomeric structure of pepsin and other aspartic proteases. The HIV-l protease subunits are 99-residue polypeptides that are homologous with the individual domains of the monomeric proteases. Structures determined by X-ray diffraction studies reveal that the active site of HIV-l protease is formed at the interface of the homodimer and consists of two aspartate residues, designated Asp and Asp one contributed by each subunit (Figure 16.29). In the homodimer, the active site is covered by two identical flaps, one from each subunit, in contrast to the monomeric aspartic proteases, which possess only a single active-site flap. 

FIGURE 16.29 (left) HIV-1 protease com-plexed with the inhibitor Crixivan (red) made by Merck. The flaps (residues 46-55 from each snbnnit) covering the active site are shown in green and the active site aspartate residues involved in catalysis are shown in white. 

This is an emerging field that has not reached its final position yet. Members of the novel class of FCP, SCP, and HAD phosphatases require Mg2+ for catalysis. An aspartate residue within the active site signature motif (DxDx(T/V)) is essential to form an acyl-phosphate intermediate. Many members of the the HAD (haloacid dehalogenase) superfamily have phosphoesterase activity [3]. Some of those protein phosphatases act on... 

If k2 > kj, the glycosyl-enzyme intermediate will accumulate, and may be trapped by the rapid denaturation of the enzyme in the presence of (saturating) amounts of substrate. With -glucoside Aj from Asp. wentii and 4-nitrophenyl [ C]-2-deoxy-) -D-irra />jo-hexopyranoside, it was possible to identify the intermediate as a glycosyl ester (acylal) of 2-deoxy-D-arabino-hexose bound to the same aspartate residue that had previously been labeled with the active-site-directed inhibitor conduritol B epoxide and with D-glucal." This constituted an important proof that the carboxylate reacting with the epoxide is directly involved in catalysis. 

In the monoamine receptors the ligand-binding domain is located within the transmembrane helices. A pocket is formed between TM3, TM5 and TM6 where the agonist binds. A conserved aspartate residue in TM3 (Asp-113 in the /(-adrenoceptor) and a... 

Figure 4. Alignment of PelZ and PelC amino acid sequences. The vertical lines indicate identical amino acids and the two points indicate homologous amino acids. The bold letters correspond to the residues probably involved in Ca + binding or catalytic function(s). The two aspartate residues probably involved in Ca binding are indicated with an asterisk. The invariant residues, probably involved in PGA cleavage, are indicated with an open circle. The folding in p-sheets is characterised by the underlined amino acids. Double underlining of PelZ residues is deduced from Chou Fasman and Robson Gamier folding predictions.

A sequence of ten amino acids (ICS-D-KTGTLT) around the phosphorylation site of Na,K-ATPase (Asp ) is highly conserved among the Na,K-, H,K-, Ca-, and Id-pumps [6]. There is also homology with the subunit of FpATP synthetase of mitochondria and chloroplasts (see [6]) except that Asp is replaced by Thr. Accordingly a covalent phosphorylated intermediate is not formed in Fi-ATPase. Mutagenesis of the phosphorylated aspartate residue in Na,K-ATPase [82], Ca-ATPase [87], or H-ATPase [88] completely blocks activity. 

In the Ca-ATPase from sarcoplasmic reticulum, oligonucleotide-directed, site-specific mutagenesis has been applied to identify amino acids involved in Ca binding. Mutation of 30 glutamate and aspartate residues, singly or in groups, in a stalk sector near the transmembrane domain has little effect on Ca " -transport. In contrast mutations to Glu ° , Glu, Asn , Thr , Asp ° or Glu ° resulted in loss... 

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. 

The pH dependence of the rate of modification shows that the pKa of Asp-32 is less than 3.165 It is seen in the high-resolution crystal structures that the carboxyl groups of the two aspartate residues are hydrogen-bonded to each other. This is similar to the ionization of maleic acid, which has pKa values of 1.9 and 6.2 (equation 16.30). 

Tao Q, Abood ME. Mutation of a highly conserved aspartate residue in... 

The inhibition of C a2+-ATPase at the active site by ATP-Im or ADP-Im with the participation of Ca2+ is illustrated by the following model. In the reaction of ATP-imidazolide with the carboxylate of Asp 351, a mixed anhydride is formed with the aspartate residue, followed by presumably nucleophilic attack of a lysine side chain, thereby displacing the nucleotide and leading to an intramolecular crosslink.[1]... 

Befort K, Tabbara L, Bausch S et al. The conserved aspartate residue in the third putative transmembrane domain of the <5-opioid receptor is not the anionic counterion for cationic opiate binding but is a constituent of the receptor binding site. Mol Pharmacol 1996 49 216-223. 

Incubation of proteins in aqueous media at elevated temperatures significantly reduces the number of methylol adducts and intermolecular cross-links. Increasing pressure not only seems to accentuate this process significantly, but also appears to be accompanied by protein cleavage at aspartate residues.13... 

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