Probing Hydrogen-Bonding Interactions in the Active Site of Medium-Chain Acyl-CoA Dehydrogenase Using Raman Spectroscopy†

Biochemistry ◽  
2003 ◽  
Vol 42 (40) ◽  
pp. 11846-11856 ◽  
Author(s):  
Jiaquan Wu ◽  
Alasdair F. Bell ◽  
Lian Luo ◽  
Avery W. Stephens ◽  
Marian T. Stankovich ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Hydrogen Bonding ◽  
Active Site ◽  
Medium Chain ◽  
Hydrogen Bonding Interactions ◽  
Chain Acyl

scholarly journals Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues†

Biochemistry ◽  
1998 ◽  
Vol 37 (41) ◽  
pp. 14605-14612 ◽  
Author(s):  
Gina J. Mancini-Samuelson ◽  
Volker Kieweg ◽  
Kim Marie Sabaj ◽  
Sandro Ghisla ◽  
Marian T. Stankovich
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Redox Properties ◽  
Amino Acid Residues ◽  
Medium Chain ◽  
Chain Acyl

scholarly journals The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

2019 ◽  
Author(s):  
Jifu Duan ◽  
Stefan Mebs ◽  
Moritz Senger ◽  
Konstantin Laun ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Proton Transfer ◽  
Active Site ◽  
Time Resolved ◽  
X Ray Crystallography ◽  
Hydrogen Bonding Interactions ◽  
Catalytic Active Site ◽  
Catalytic Intermediates ◽  
Time Resolved Infrared Spectroscopy

The H2 conversion and CO inhibition reactivity of nine [FeFe]-hydrogenase constructs with semi-artificial cofactors was studied by in situ and time-resolved infrared spectroscopy, X-ray crystallography, and theoretical methods. Impaired hydrogen turnover and proton transfer as well as characteristic CO inhibition/ reactivation kinetics are assigned to varying degrees of hydrogen-bonding interactions at the active site. We show that the probability to adopt catalytic intermediates is modulated by intramolecular and protein-cofactor interactions that govern structural dynamics at the active site of [FeFe]-hydrogenases.<br>

scholarly journals A crystallographic study of the Toho-1 β-lactamase acylation mechanism

2014 ◽  
Vol 70 (a1) ◽  
pp. C1207-C1207
Author(s):  
Leighton Coates
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Ligand Complex ◽  
Transition State Analog ◽  
Hydrogen Bonding Interactions ◽  
Active Site Region ◽  
Neutron Crystallography

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

scholarly journals The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

2019 ◽  
Author(s):  
Jifu Duan ◽  
Stefan Mebs ◽  
Moritz Senger ◽  
Konstantin Laun ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Proton Transfer ◽  
Active Site ◽  
Time Resolved ◽  
X Ray Crystallography ◽  
Hydrogen Bonding Interactions ◽  
Catalytic Active Site ◽  
Catalytic Intermediates ◽  
Time Resolved Infrared Spectroscopy

The H2 conversion and CO inhibition reactivity of nine [FeFe]-hydrogenase constructs with semi-artificial cofactors was studied by in situ and time-resolved infrared spectroscopy, X-ray crystallography, and theoretical methods. Impaired hydrogen turnover and proton transfer as well as characteristic CO inhibition/ reactivation kinetics are assigned to varying degrees of hydrogen-bonding interactions at the active site. We show that the probability to adopt catalytic intermediates is modulated by intramolecular and protein-cofactor interactions that govern structural dynamics at the active site of [FeFe]-hydrogenases.<br>

scholarly journals Xanthine oxidase–product complexes probe the importance of substrate/product orientation along the reaction coordinate

2017 ◽  
Vol 46 (39) ◽  
pp. 13242-13250 ◽  
Author(s):  
Jing Yang ◽  
Chao Dong ◽  
Martin L. Kirk
Keyword(s):  
Raman Spectroscopy ◽  
Hydrogen Bonding ◽  
Xanthine Oxidase ◽  
Resonance Raman Spectroscopy ◽  
Resonance Raman ◽  
Reaction Coordinate ◽  
Substrate Orientation ◽  
Hydrogen Bonding Interactions

Resonance Raman spectroscopy has been used to probe substrate orientation and hydrogen bonding interactions in a xanthine oxidase catalytic intermediate.

Ring Current Effects in the Active Site of Medium-Chain Acyl-CoA Dehydrogenase Revealed by NMR Spectroscopy

2005 ◽  
Vol 127 (23) ◽  
pp. 8424-8432 ◽  
Author(s):  
Jiaquan Wu ◽  
Alasdair F. Bell ◽  
Andrew A. Jaye ◽  
Peter J. Tonge
Keyword(s):  
Nmr Spectroscopy ◽  
Active Site ◽  
Ring Current ◽  
Medium Chain ◽  
Chain Acyl

scholarly journals Biochemical characterization of a variant human medium-chain acyl-CoA dehydrogenase with a disease-associated mutation localized in the active site

1999 ◽  
Vol 337 (2) ◽  
pp. 225-230 ◽  
Author(s):  
Burkhard KÜCHLER ◽  
Abdel-Ghany ABDEL-GHANY ◽  
Peter BROSS ◽  
Andreas NANDY ◽  
Ihab RASCHED ◽  
...  
Keyword(s):  
Active Site ◽  
Biochemical Characterization ◽  
Genetic Disorder ◽  
Ph Dependence ◽  
Flow Measurements ◽  
Medium Chain ◽  
Prevalent Disease ◽  
Chain Acyl ◽  
Electron Transferring Flavoprotein

Medium-chain acyl-CoA dehydrogenase (MCADH) deficiency, an autosomal recessive inherited disorder, is the most common genetic disorder in mitochondrial β-oxidation in humans. In addition to one prevalent disease-causing mutation (K304E), a series of rarer mutations has been reported, but none of these has yet been characterized in detail. We report here on the biochemical characterization of the purified recombinant mutant protein in which threonine is replaced by alanine at position 168 of the mature protein (T168A-MCADH). It is the first mutation to be found in patients that is located in the active site of the enzyme. Thr-168 is hydrogen-bonded to the flavin N(5) of the cofactor FAD. The thermostability of T168A-MCADH is markedly decreased compared with human wild-type MCADH (hwt-MCADH). Catalytic activity with ferricenium as acceptor is lowered by 80% and with the natural acceptor electron-transferring flavoprotein by over 90% compared with hwt-MCADH. In the mutant the extent of flavin semiquinone formed on reduction is approx. 50% that of hwt-MCADH. The pK reflected by the pH-dependence of Vmax is shifted from approx. 8.2 (hwt-MCADH) to approx. 7 (T168A-MCADH) and the rates of enzyme flavin reduction (stopped-flow measurements) are only approx. 1/10 those of the parent enzyme. These properties are discussed in the light of the possible mechanisms leading to disease in humans.

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