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Enzyme Catalysis
Published in Harvey W. Blanch, Douglas S. Clark, Biochemical Engineering, 1997
Harvey W. Blanch, Douglas S. Clark
In metalloenzymes a metal ion is present at the active site and this ion plays an important role in stabilizing negative charges that are formed in electrophilic catalysis. Zinc, copper and cobalt are commonly involved in coordination of oxyanions involved as reaction intermediates. The enzyme carboxypeptidase-A, which is a carboxyl-terminus exopeptidase (i.e., it acts by hydrolyzing the peptide from the carboxylic acid terminus), contains Zn2+ which polarizes the carbonyl oxygen of the terminal peptide bond. The terminal carboxylate is charge paired with the guanidinium cation of Arg145, leading to polarization of the terminal carboxylic carbonyl group. This polarization increases the electrophilicity of the carbonyl carbon and facilitates nucleophile-mediated hydrolysis of the amide bond. This is illustrated in the accompanying figure. In addition to stabilizing negative charges, metal ions serve as a source of potent nucleophilic hydroxyl ions. Metal-bound water molecules provide these nucleophilic hydroxyl groups at neutral pH.
Product Quality and Process
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Other amino acid sequence variants may occur post-translationally. At a low frequency, the leader sequence that directs the nascent protein to translocate into the endoplasmic reticulum may not be cleaved, producing a variant. Many proteins have lysine or arginine in their C-terminus. The basic amino acids in the C-terminus are liable to cleavage by carboxypeptidase. The cleavage of lysine at the C-terminus of the heavy chain of many IgG molecules by carboxypeptidase is often not complete, leaving some molecules with uncleaved C-terminus lysine.
Quality and lifecycle management
Published in Sarfaraz K. Niazi, Biosimilars and Interchangeable Biologics, 2016
Amino acid sequencing is divided into N- and C-terminal sequencing. Amino terminal (N-terminal) sequence analysis is based on the modification of the unmodified N-terminal amino group of the peptide/protein with phenylisothiocyanate (PITC) followed by acid cleavage of the peptide bond releasing the phenylthiohydantoin (PTH) derivative of the amino acid. A new amino group of the next amino acid is now available to react with PITC and the protein sequencing will thus take place in a cyclic manner (Edman degradation). The resulting PTH-amino acid derivative is thereafter analyzed on a PTH amino acid analyzer based on HP-RPC separation technology. The cyclic reaction is not 100% effective and in practice from 20 to 40 amino acid residues can be sequenced by the method described in the low picomol range. Entire sequences may be obtained by cleavage of the protein with specific proteases such as trypsin, V8 protease, or chymotrypsin followed by HP-RPC separation, MALDI mass spectroscopy (MALDI-MS), and Edman sequencing of the peptides separated. Carboxy-terminal (C-terminal) sequence analysis is used for confirmation of the C-terminal part of the peptide or protein. Typically, the peptide/protein is digested with enzymes such as carboxypeptidase A, P, or Y, which are exopeptidases removing one amino acid residue, one at a time, from the C-terminus of the peptide/protein. The enzymes have different specificity and mixtures may be used. After digestion, the released amino acids are removed from the residual peptide/protein and analyzed by amino acid analysis. The reaction is followed over time allowing for determination relative amounts released. Recently an automated procedure has been introduced consisting of two principal reaction events. First, the d-carboxylic acid group of the C-terminal amino acid residue is activated with trifluoroacetic acid (TFA) and coupled with diphenyl phosphoroisothiocyanatidate (DPP-ITC) in the presence of pyridine. The resulting peptide/protein thiohydantoin is then cleaved with potassium trimethylsilanolate to release the thiohydantoin amino acid from the now shortened peptide/protein. The thiohydantoin derivative is identified by RP-HPC analysis at 269 nm. The method is currently able to provide three to five cycles of sequence information. The range of sensitivity is from 10 to 20 pmol of protein Bicinchoninic assay
Prostate-specific membrane antigen-directed imaging and radioguided surgery with single-photon emission computed tomography: state of the art and future outlook
Published in Expert Review of Medical Devices, 2022
Luca Filippi, Barbara Palumbo, Viviana Frantellizzi, Susanna Nuvoli, Giuseppe De Vincentis, Angela Spanu, Orazio Schillaci
PSMA is a zinc-containing metal-enzyme transmembrane glycoprotein of type II, that is intensely up-regulated in PC cells with a grade of expression strictly correlated with tumor stage and biological aggressiveness. PSMA’s exact role in prostate normal tissue is still unclear, although it has been hypothesized it might have a trophic function. In past years 111In-capromab pendetide, a monoclonal antibody targeting PSMA, was used for the imaging of PC recurrence and metastases, but it showed limited sensitivity and specificity [32], since this radiocompound recognizes the PSMA-intracytoplasmatic domain that is available only in necrotic or dying tumor cells. Worthy of note, glutamate carboxypeptidase activity is localized in PSMA extracellular domain, thus being exposed to the extracellular space and particularly suitable for theranostic approaches [33].
A review on bio-functional models of catechol oxidase probed by less explored first row transition metals
Published in Journal of Coordination Chemistry, 2022
Rashmi Rekha Tripathy, Shuvendu Singha, Sohini Sarkar
Among all transition metal ions, zinc ion possesses much biological relevance in living systems. Important metalloenzymes, such as aminopeptidase, carboxypeptidase, alcohol dehydrogenase, and carbonic anhydrase, contain zinc(II) as cofactor which serves an essential role to activate catalytic features of them. All these enzymes play significant parts in cellular functions occurring in plants as well as in animal bodies. Designing new zinc(II) complexes to check their viability to biomimic active sites, which are present in different metalloenzymes, is an attractive field of modern research. Searching for new functional models for catechol oxidase has raised interest in the research of bioinorganic chemistry. Zinc(II)-complexes have been found to be capable of catechol oxidase activity. Although it is believed that the redox innocent nature of zinc cannot permit metal centric oxidation of catechols, quite satisfactory turnover numbers obtained for some of the zinc(II)-complexes confirm ligand centric mechanism like nickel(II).
Digital PET and detection of recurrent prostate cancer: what have we gained, and what is still missing?
Published in Expert Review of Medical Devices, 2021
Luca Filippi, Orazio Schillaci
Positron emission computed tomography (PET/CT) with radiopharmaceuticals binding to prostate-specific membrane antigen (PSMA) has recently emerged as a powerful tool for the detection of recurrent prostate cancer (PCa) [1]. PSMA is a transmembrane type II glycoprotein, characterized by an enzymatic glutamate carboxypeptidase activity, overexpressed by prostate adenocarcinoma, but only minimally detectable in prostate normal tissue [2]. Several efforts have been made for the development of radiopharmaceuticals capable of binding to PSMA. In this regard, a turning point has been represented by the introduction of small compounds linked to glutamate isostere or glutamate (i.e. PSMA inhibitors), which bind to PSMA extracellular domain exploiting its enzymatic activity. Among PSMA inhibitors, 68Ga-PSMA-11 has been recently approved by the Food and Drug Administration (FDA) as the first drug for the clinical imaging of men affected by PCa [3].