Abstract:PHM is a dicopper enzyme that plays a vital role in the amidation of glycine extended pro-peptides. One of the crucial aspects of its chemistry is the transfer of two electrons from an electron-storing and transferring site (CuH) to the oxygen binding site and catalytic center (CuM) over a distance of 11 Å during one catalytic turnover event. Here we present our studies on the first electron transfer step (reductive phase) in the WT PHM as well as its variants. Stopped-flow was used to record the reduction kin… Show more
“…Notably, copper ions are commonly found as the active sites in natural enzymes, such as laccase and amine oxidases. In this context, excellent works by Lu and co‐workers showed that copper is the element of choice for the design and synthesis of enzymes; however, the skeleton of these modified Cu‐containing enzymes still consists of proteins or amino acids. Thus, the development of new artificial protein‐like ligands containing anchored copper ions to mimic the activity of native enzymes is of great interest.…”
Section: Introductionmentioning
confidence: 80%
“…In this context,e xcellent works by Lu [20][21][22][23] and co-workerss howed that copperi st he element of choice for the design and synthesis of enzymes; however,t he skeleton of these modified Cu-containing enzymes still consists of proteins or amino acids. In this context,e xcellent works by Lu [20][21][22][23] and co-workerss howed that copperi st he element of choice for the design and synthesis of enzymes; however,t he skeleton of these modified Cu-containing enzymes still consists of proteins or amino acids.…”
Artificial enzymes is an emerging field of research owing to the remarkable advantages of enzyme mimics over their natural counterpart, including tunable catalytic efficiencies, lower cost, ease of preparation, and excellent tolerance to variations of the reaction system. Herein, we report an efficient peroxidase mimic based on a copper-modified covalent triazine framework (CCTF). Owing to its unique specific surface area, atomically dispersed active Cu sites, efficient electron transfer, and enhanced photo-assisted enzyme-like activity, the CCTF showed enhanced peroxidase-like enzyme activity. Therefore, copper modification represents an effective route to tailor the peroxidase-like activity of the covalent triazine frameworks. Furthermore, the mechanism of the enhanced peroxidase-like activity and stability of the CCTF were investigated. As a proof of concept, the CCTF was used for the colorimetric detection of H O and decomposition of organic pollutants. This work provides a new strategy for the design of enzyme mimics with a broad range of potential applications.
“…Notably, copper ions are commonly found as the active sites in natural enzymes, such as laccase and amine oxidases. In this context, excellent works by Lu and co‐workers showed that copper is the element of choice for the design and synthesis of enzymes; however, the skeleton of these modified Cu‐containing enzymes still consists of proteins or amino acids. Thus, the development of new artificial protein‐like ligands containing anchored copper ions to mimic the activity of native enzymes is of great interest.…”
Section: Introductionmentioning
confidence: 80%
“…In this context,e xcellent works by Lu [20][21][22][23] and co-workerss howed that copperi st he element of choice for the design and synthesis of enzymes; however,t he skeleton of these modified Cu-containing enzymes still consists of proteins or amino acids. In this context,e xcellent works by Lu [20][21][22][23] and co-workerss howed that copperi st he element of choice for the design and synthesis of enzymes; however,t he skeleton of these modified Cu-containing enzymes still consists of proteins or amino acids.…”
Artificial enzymes is an emerging field of research owing to the remarkable advantages of enzyme mimics over their natural counterpart, including tunable catalytic efficiencies, lower cost, ease of preparation, and excellent tolerance to variations of the reaction system. Herein, we report an efficient peroxidase mimic based on a copper-modified covalent triazine framework (CCTF). Owing to its unique specific surface area, atomically dispersed active Cu sites, efficient electron transfer, and enhanced photo-assisted enzyme-like activity, the CCTF showed enhanced peroxidase-like enzyme activity. Therefore, copper modification represents an effective route to tailor the peroxidase-like activity of the covalent triazine frameworks. Furthermore, the mechanism of the enhanced peroxidase-like activity and stability of the CCTF were investigated. As a proof of concept, the CCTF was used for the colorimetric detection of H O and decomposition of organic pollutants. This work provides a new strategy for the design of enzyme mimics with a broad range of potential applications.
“…The data were fit by non-linear regression to a rate equation which describes the process as a combination of fast and slow pseudo first order reactions and is identical to that used previously to analyze the DMPD reduction of the WT PHM. 81 A t = A 0 + A 1 1 − e −k 1…”
Section: Reduction By Nn'-dimethyl Phenylenediamine (Dmpd)mentioning
Mononuclear copper monooxygenases peptidylglycine monooxygenase (PHM) and dopamine βmonooxygenase (DBM) catalyze the hydroxylation of high energy C-H bonds utilizing a pair of chemically distinct copper sites (CuH and CuM) separated by 11 Å. In earlier work, we constructed single-site PHM variants that were designed to allow study of the M-and H-centers independently in order to place their reactivity sequentially along the catalytic pathway. More recent crystallographic studies suggest that these single-site variants may not be truly representative of the individual active sites. In this work we describe an alternative approach that uses rational design to construct an artificial PHM model in a small metallochaperone scaffold. Using site-directed mutagenesis, we constructed variants that provide a His 2 Met copper-binding ligand set that mimics the M-center of PHM. The results show that the model accurately reproduces the chemical and spectroscopic properties of the M-center, including details of the methionine coordination, and the properties of Cu(I) and Cu(II) states in the presence of endogenous ligands such as CO and azide. The rate of reduction of the Cu(II) form of the model by the chromophoric reductant N,N'-dimethyl phenylenediamine (DMPD) has been compared with that of the PHM M-center, and the reaction chemistry of the Cu(I) forms with molecular oxygen has also been explored, revealing an unusually low reactivity towards molecular oxygen. This latter finding emphasizes the importance of substrate triggering of oxygen reactivity, and implies that the His 2 Met ligand set while necessary, is insufficient on its own to activate oxygen in these enzyme systems.
“…The structures of the PHM and PAL domains of rat PAM, alone and in complexes with substrates, inhibitors, and other ligands, have been determined 11 – 15 . Other techniques used to study PHM have ranged from kinetics and kinetic isotope effect measurements 16 – 20 , to inhibitor design 21 – 24 , spectroscopy 25 – 29 including X-ray absorption spectroscopy (XAS) 17 , 30 – 33 , and computational studies 11 , 34 – 36 . These studies have provided significant insights into the mechanism of both domains, especially of PHM.…”
The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role.
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