Amidating enzyme

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. 

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. 

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 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... 

The PHM domain in peptidylglycine a-amidating enzyme shows 28% sequence identity to part of the amino acid sequence in D/3M. As described below, the spectroscopic and redox properties of the copper ions in D/3M and PHM are also highly similar. Hence, although crystallographic data are only available for PHM, the active site structures and catalytic mechanisms of D/3M and PHM are assumed to be similar. For this reason, they will be discussed together. 

Boswell JS, Reedy BJ, Kulathila R, Merkler D, Blackburn NJ. 1996. Structural investigations on the coordination environment of the active site copper centers of recombinant bifunctional peptidylglycine a-amidating enzyme. Biochemistry 35 12241-12250. 

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