scholarly journals A Kinetic Approach to Defining the Role of Chemically Modifiable Residues at the Active Sites of Enzymes

1975 ◽  
Vol 28 (4) ◽  
pp. 379
Author(s):  
Leonie K Ashman ◽  
D Bruce Keech
Keyword(s):  
Biological Activity ◽  
Catalytic Activity ◽  
Kinetic Analysis ◽  
Active Site ◽  
Initial Velocity ◽  
Active Sites ◽  
Kinetic Approach ◽  
Modified Enzyme ◽  
Enzyme Molecules

Initial velocity studies can be used to define the function of chemically modifiable residues in the active site of a multisubstrate enzyme. Since the method relies on measuring biological activity, it has the advantage that it can be used with small amounts of relatively impure enzyme, but requires that modified enzyme molecules have some residual catalytic activity. The kinetic analysis of the modified enzyme can be carried out in the presence of some unmodified enzyme molecules.

scholarly journals Insights into the role of an Fe–N active site in the oxygen reduction reaction on carbon-supported supramolecular catalysts

RSC Advances ◽  
2020 ◽  
Vol 10 (15) ◽  
pp. 8709-8716
Author(s):  
Lin Gu ◽  
Yunyun Dong ◽  
Yan Zhang ◽  
Bo Wang ◽  
Qing Yuan ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Oxygen Reduction Reaction ◽  
Oxygen Reduction ◽  
Active Site ◽  
Active Sites ◽  
Reduction Reaction ◽  
Alkaline Media ◽  
Supramolecular Catalysts

The PPYTZ–Fe/C catalyst containing Fe–N active sites exhibited high ORR catalytic activity and stability in alkaline media with a four-electron pathway progress.

On the role of lysine in the active site Ser-X-X-Lys region of penicillin-recognizing enzymes

2010 ◽  
Vol 88 (1) ◽  
pp. 1-4
Author(s):  
Saul Wolfe ◽  
Kiyull Yang
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Density Functional ◽  
Basis Set ◽  
Penicillin G ◽  
Hydroxyl Group ◽  
Functional Theory ◽  
One Step ◽  
Optimized Geometries

Using Autodock, docking of penicillin G to the crystal structures of penicillin-recognizing enzymes leads to an alignment in the active site Ser-X-X-Lys region consisting of the serine hydroxyl group, the terminal amino group of lysine, a second hydroxyl group, and the N–C=O of the β-lactam. This alignment is consistent with the notion that acylation of the serine hydroxyl group proceeds by a one-step cooperative mechanism in which C–O bond formation and proton transfer to the β-lactam nitrogen take place through a heteroatom bridge. For the cooperative ring opening of penam by two molecules of methanol and one molecule of methylamine or one molecule of water, density functional theory with the B3LYP DFT gradient-corrected functional and the 6–31G(d) basis set reproduces the alignment seen in the docked structures. Methylamine lowers the barrier calculated at MP2/6–31G(d) from the DFT-optimized geometries by 3 kcal/mol; water increases the barrier by 4 kcal/mol. The function of the conserved lysine in the active sites of penicillin-recognizing enzymes is therefore to catalyze the formation of an acyl enzyme by a cooperative mechanism.

scholarly journals Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

2015 ◽  
Vol 60 (2) ◽  
pp. 990-1002 ◽  
Author(s):  
Susann Skagseth ◽  
Trine Josefine Carlsen ◽  
Gro Elin Kjæreng Bjerga ◽  
James Spencer ◽  
Ørjan Samuelsen ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Point Mutations ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
Hydrophilic Residues

ABSTRACTMetallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeledin silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family

2020 ◽  
Vol 168 (2) ◽  
pp. 191-202
Author(s):  
Michael R Duff ◽  
Jasmina S Redzic ◽  
Lucas P Ryan ◽  
Natasia Paukovich ◽  
Rui Zhao ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Biliverdin Reductase ◽  
X Ray Crystallography ◽  
Functional Consequences ◽  
Dynamics And Function ◽  
And Function ◽  
Slow Turnover ◽  
Binding Substrate

Abstract Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme ‘clamps’ formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.

Catalytic mechanisms of oxygen-containing groups over vanadium active sites in an Al-MCM-41 framework for production of 2,5-diformylfuran from 5-hydroxymethylfurfural

2020 ◽  
Vol 10 (1) ◽  
pp. 278-290 ◽  
Author(s):  
Li-Juan Liu ◽  
Zhao-Meng Wang ◽  
Ya-Jing Lyu ◽  
Jin-Feng Zhang ◽  
Zhou Huang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Active Sites ◽  
Hydroxyl Group ◽  
Catalytic Mechanisms ◽  
Mcm 41

In the V-doped Al-MCM-41 framework, the [V-1] active site with a hydroxyl group displays better catalytic activity than the [V-0] active site without a hydroxyl group toward the oxidation of 5-hydroxymethylfurfural to 2,5-diformylfuran.

scholarly journals Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

2015 ◽  
Vol 81 (7) ◽  
pp. 2612-2624 ◽  
Author(s):  
Elena Sugrue ◽  
Nicholas J. Fraser ◽  
Davis H. Hopkins ◽  
Paul D. Carr ◽  
Jeevan L. Khurana ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Evolutionary Transition ◽  
Functional Changes ◽  
Metal Ion Analysis ◽  
Amidohydrolase Superfamily ◽  
Kinetics Of ◽  
Hydrolysis Of

ABSTRACTThe amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.

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