Enzymes amidating enzyme

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

Lipoic acid exists as a mixture of two structures a closed-ring disulfide form and an open-chain reduced form (Figure 18.33). Oxidation-reduction cycles interconvert these two species. As is the case for biotin, lipoic acid does not often occur free in nature, but rather is covalently attached in amide linkage with lysine residues on enzymes. The enzyme that catalyzes the formation of the lipoamide nk.2Lg c requires ATP and produces lipoamide-enzyme conjugates, AMP, and pyrophosphate as products of the reaction. 

Guo, X.-L., Deng, G., Xu, J. and Wang, M.-X. (2006) Immobilization of Rhodococcus sp. AJ270 in alginate capsules and its application in enantioselective biotransformation of ira/i.s-2-methyl-3- phenyl-oxiranecarbonitrile and amide. Enzyme and Microbial Technology, 39, 1-5. 

Note that penicillins and structurally related antibiotics are frequently deactivated by the action of bacterial -lactamase enzymes. These enzymes also contain a serine residue in the active site, and this is the nucleophile that attacks and cleaves the P-lactam ring (see Box 7.20). The P-lactam (amide) linkage is hydrolysed, and then the inactivated penicillin derivative is released from the enzyme by further hydrolysis of the ester linkage, restoring the functional enzyme. The mode of action of these enzymes thus closely resembles that of the serine proteases there is further discussion in Box 7.20. 

Peptidylglycine monooxygenase [EC 1.14.17.3], also known as peptidyl a-amidating enzyme and peptidylglycine 2-hydroxylase, catalyzes the reaction of a peptidylglycine with ascorbate and dioxygen to produce a pepti-dyl(2-hydroxyglycine), dehydroascorbate, and water. 

A drawback of all of these methods is that they produce racemic amino acids. If the product is to be used in place of a natural amino acid, it must first be resolved. This can be accomplished by the traditional method of preparing and separating diastereomeric salts. Alternatively, nature s help can be enlisted through the use of enzymes. In one method the racemic amino acid is converted to its amide by reaction with acetic anhydride. The racemic amide is then treated with a deacylase enzyme. This enzyme catalyzes the hydrolysis of the amide back to the amino acid. However, the enzyme reacts only with the amide of the naturally occurring L-amino acid. The L-amino acid is easily separated from the unhydrolyzed D-amide. The following equation illustrates the use of this process to resolve methionine ... 

I. A. Yamskov, T. V. Tichonova, V. A. Davankov, Pronase-catalyzed hydrolysis of amino acid amides, Enzyme Microb. Tech., 8 (1986), 241-244. 

A number of copper requiring enzymes are located at the cell surface or are exported into the extracellular milieu. Examples of such secretory Cu-enzymes include copper requiring ferroxidases that fimction in iron transport (e g. ceruloplasmin, CP), enzymes for neurotransmission (peptidyl amidating enzyme and dopamine hydroxylase), an extracellular superoxide dismutase (SOD) that fimctions in antioxidant defense and enzymes for formation of connective tissue (lysyl oxidase), and pigments (tyrosinase) (reviewed in ). En route to their designated location, each of these enzymes passes through a specialized compartment of the late Golgi where copper insertion takes place. 

FIGURE 9-86 Ascorbate is required for the activity of dopamine hydrosylase, also called dopamine-p-monooKygenase. The mechanism of the reaction is quite similar to that of amidating enzyme, an ascorbate-requiring enzyme that catalyzes the hydroxylation of polypeptides, during the course of a two-step sequence. 

Amidating enzyme is a copper metalloeruiyme that requires both oxygen and ascorbic add. One copper atom is bound to three residues of histidine (his 107,108, and 172). The other copper atom is bound to two residues of histidine (his 242 and 244), and to a residue of methionine (met 314). The enzyme is actually bifunctionai, that is, it consists of two separate enzymes occurring in a single polypeptide chain. The two enzymes work, one after the other, to create tlte amidated polypeptide (Prigge et ai 1997),... 

Mild oxidation of the C-terminus of a peptide can bring about the formation of an acylimide, which is then easily hydrolysed. This change, which is thought to involve oxidative a-hydroxylation of the C-terminal amino acid residue (or oxidation of the oxazolone formed from the C-terminal amino acid residue) has been accomplished under physiological conditions in the absence of enzymes when the C-terminus is activated as an anhydride or as an oxazolone (the reactions are shown in Scheme 8.3 Barrett et al., 1978) and the search for an enzymic equivalent has led to the discovery of a family of amidating enzymes. This, then, is how a biologically inactive propeptide , with one amino-acid residue more than the peptide amide into which it is processed, is a latent precursor for many hormones that are peptide amides (calcitonin, vasopressin, etc.). 

FIGURE 10.38 Distribution of markers in pancreatic endocrine tumors. SYN, synaptophysin NSE, neuron-specific enolase LMWCK, low-molecular-weight cytokeratin CgA, chromogranin A PC2, pro-convertase 2 PCM, peptidylglycine alpha-amidating enzyme PC3, proconvertase 3 NFP, neurofilament protein HCC(a), human chorionic gonadotropin alpha VIM, vimentin. 

Kimura N, Pilichowska M, Okamoto H, et al. Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors. Mod Pathol. 2000 13 140-146. 

Francisco, W. A., Merkler, D. J., Blackburn, N. J., Klinman, J. P. (1998) Kinetic mechanism and intrinsic isotope effects for the peptidylglycine a-amidating enzyme reaction. Biochemistry 37, 8244-8252. 

A pantothenic acid hydrolase (pantothenase) activity has been isolated from Pseudomonas fluorescens and other Pseudomonas strains. This enzyme hydrolyzes the amide bond of pantothenic acid 2 to form pantoic acid 5 (or pantoyl lactone) and /i-alanine 7 (EC 3.5.1.22) (Equation (10)). A detailed kinetic study of the reaction mechanism has shown that the reaction is partially reversible because of the formation of an acyl—enzyme (pantoyl-enzyme) intermediate during the course of catalysis, which may react with either water or / -alanine to form pantoic acid (the product hydrolysis) or pantothenic acid (the original substrate) Such a mechanism suggests that this enzyme could act as a pantothenate synthase, as reaction of the active site serine with pantoyl lactone would result in the formation of the pantoyl—enzyme intermediate. However, no biochemical or genetic evidence is currently available to support such a hypothesis. 

A similar copper-dependent hydroxylase constitutes the N-terminal domain of the peptidylglycine a-amidating enzyme (Eq. 10-11). This bifunctional enzyme hydroxylates C-terminal glycines in a group of neuropeptide hormones and other secreted peptides. The second functional domain of the enzyme cleaves the hydroxylated glycine to form a C-terminal... 

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