Peptide amidase

Cysteine string protein (CSP) Cytochrome b561 Peripheral membrane protein that is paimitoylated on >10 cysteines. May have a role in Ca2+ sensitivity of exocytosis. Electron-transport protein required for intravesicular monooxygenases in subsets of secretory vesicles. Required for dopamine- -hydroxylase and peptide amidase activity. 

Valina ALB, Mazumder-Shivakumar D, Bruice T C (2004) Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase computational studies of the ground state, transition state, and intermediate. Biochemistry 43 15657-15672... 

SteUces-Ritter, U., Wyzgol, K., and Kula, M. 1995. Purification and characterization of a newly screened microbial peptide amidase. Applied Microbiology and Biotechnology, 44 393-8. 

The additional opportunity to hydrolyze selectively C-terminal peptide amides is of particular interest. For example, a peptide amidase from the flavedo of oranges has broad substrate specificity and accepts Boc-, Trt-, Z-, and Bz-protected and N-terminal unprotected peptide amides (Scheme The C-terminal amides are saponified by this biocatalyst in high yields... 

The enz5une preparation was obtained from the flavedo of oranges, that is, from the outmost colored layer of the peel, by a two-step purification involving fractionating salt precipitation and ion-exchange chromatography. The peptide amidase had an activity of 2 to 100 U-mg , depending on the substrate, where an activity of 1 U corresponds to the formation of 1 pmol of product per minute at 30 °C and... 

An instructive example of the occurrence of selectivity problems is available from the field of enzymatic peptide synthesis. For instance, the following complex reaction scheme shows where parallel reactions and consecutive reactions occur. It describes the synthesis of Tyr-Arg from the electrophile Tyr-OEt and the nucleophile Arg-NH2 by simultaneous use of Carboxypeptidase Y (CPD-Y) and Peptide amidase (PA) from orange flavedo[54) (Eq. (11)). 

The protease Carboxypeptidase Y (CPD-Y) reacts with the tyrosine ester forming an acylenzyme activated complex, which is further converted in a competitive way. Aminolysis of the acylenzyme by Arg-NH2 (2) yields the dipeptide amide Tyr-Arg-NH2. The competing hydrolysis (1) yields the non-activated tyrosine. The dipeptide amide Tyr-Arg-NH2 is deamidated selectively by peptide amidase (PA) to the desired... 

Now, however this situation has changed. Steinke and Kula have isolated an unusual peptide amidase from orange flavedo, which is very selective for the... 

Peptide Amidase Origin Citrus sinensis Fluka... 

More than 100 members of the AS family of enzymes have been reported in the literature, but only for malonamidase (MAE2) (Shin et al., 2002) and C-terminal peptide amidase (PAM) (Labahn et al., 2002), two soluble bacterial enzymes, structural data are available. All three resolved structures of AS enzymes (FAAH, MAE2, and PAM) revealed a common core, consisting of a twisted p-sheet of 11 mixed strands, surrounded by a large number of a-helices (those of FAAH are shown in Fig. 4.3). Compared to other AS enzymes, which are mostly soluble proteins, FAAH displays two distinguished features i) integration into membranes, and ii) strong preference for hydrophobic substrates. 

Interesting new enzymes can of course also be isolated from non-microbial sources. A recent example of a useful novel enzyme or plant origin is the peptide amidase from orange flavedo (Fig. 5) discovered by Steinke and Kula [130, 131]. This enzyme, which has an extremely wide substrate range, is useful for C-terminal enzymatic deprotection in peptide synthesis under very mild conditions. Substrates of this peptide amidase are protected and unprotected peptide amides, N-protected amino acid amides. The enzyme is stereoselective with regard to the C-terminal position only L-amino acid amides are accepted as substrates, with the exception of proline. Other interesting enzymes of plant origin are the oxynitrilases (see below). 

Figure 5. Peptide amidase for the C-terminal peptide deprotection under mild conditions (selected examples).

Cerovsky, V., Kula, M.-R. (2001). Studies on peptide amidase-catalysed C-terminal peptide amidation in organic media with respect to its substrate specificity. BiotechnoL Appl. Biochem., 33,183-187. 

Arif, M. I., Toplak, A., Szymanski, W., Feringa, B. L., Nuijens, T., Quaedflieg, P. J. L. M., Wu, B., Janssen, D. B. (2014). One-step C -terminal deprotection and activation of peptides with peptide amidase from Stenotrophomonas mdtophilia in neat organic solvent. Adv. Synth. Catal, 356,2197-2202. 

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