scholarly journals Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole

2010 ◽  
Vol 107 (5) ◽  
pp. 1960-1965 ◽  
Author(s):  
Daniel A. Kraut ◽  
Paul A. Sigala ◽  
Timothy D. Fenn ◽  
Daniel Herschlag
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Ketosteroid Isomerase ◽  
X Ray ◽  
Enzyme Active Site ◽  
Hydrogen Bonding Interactions ◽  
Rate Effects ◽  
Paradoxical Effects ◽  
Moderate Rate

The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 105-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a less-severe 102-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-Å resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-Å distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and site-directed mutagenesis experiments.

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3028-3034 ◽  
Author(s):  
Soohee Lee ◽  
Asim K. Debnath ◽  
Colvin M. Redman
Keyword(s):  
Amino Acids ◽  
Blood Group ◽  
Active Site ◽  
Neutral Endopeptidase ◽  
Site Directed Mutagenesis ◽  
Peptide Hydrolysis ◽  
Enzyme Active Site ◽  
Endopeptidase 24.11 ◽  
Outer Domain ◽  
Group Protein

Abstract In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3–converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of α-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.

scholarly journals Albino mutants of Streptomyces glaucescens tyrosinase

1991 ◽  
Vol 274 (3) ◽  
pp. 707-713 ◽  
Author(s):  
M P Jackman ◽  
A Hajnal ◽  
K Lerch
Keyword(s):  
Active Site ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Carbonyl Derivatives ◽  
Hydrogen Bonding Interactions ◽  
Albino Phenotype ◽  
Derivatives Of ◽  
Streptomyces Glaucescens

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

scholarly journals Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

2013 ◽  
Vol 110 (28) ◽  
pp. E2552-E2561 ◽  
Author(s):  
P. A. Sigala ◽  
A. T. Fafarman ◽  
J. P. Schwans ◽  
S. D. Fried ◽  
T. D. Fenn ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Proton Transfer ◽  
Active Site ◽  
Ketosteroid Isomerase

scholarly journals An Investigation of Five Component [3+2] Self-Assembled Cage Formation Using amidinium...carboxylate Hydrogen Bonds

2021 ◽  
Author(s):  
Chriso Thomas ◽  
Emer Foyle ◽  
Samuel Walker ◽  
Nicholas White
Keyword(s):  
Hydrogen Bond ◽  
Self Assembly ◽  
Solvent Mixtures ◽  
Hydrogen Bond Donor ◽  
Cage Structure ◽  
Hydrogen Bond Acceptor ◽  
X Ray ◽  
Hydrogen Bonding Interactions ◽  
The Individual ◽  
And Diffusion

The assembly of hydrogen bonded cages using amidinium∙∙∙carboxylate hydrogen bonding interactions was investigated. A new tris-amidinium hydrogen bond donor tecton based on a tetraphenylmethane scaffold was prepared and its self–assembly with the terephthalate anion studied, and a new tricarboxylate hydrogen bond acceptor tecton was synthesized and its assembly with the 1,3-benzenebis(amidinium) hydrogen bond donor explored. In both cases, molecular modelling indicated that the formation of the cages was geometrically feasible and 1H NMR spectroscopic evidence was consistent with interactions between the components in competitive d6- DMSO solvent mixtures. DOSY NMR spectroscopy of both systems indicated that both components diffuse at the same rate as each other, and diffusion coefficients were consistent with cage formation, and with the formation of assemblies significantly larger than the individual components. An X-ray crystal structure showed that one of the assemblies did not have the desired cage structure in the solid state

Correspondence: Hydrogen Bond to Gold? 1H NMR is Not a Proof of Hydrogen Bonds in Transition Metal Complexes

2018 ◽  
Author(s):  
Jan Vícha ◽  
Cina Foroutan-Nejad ◽  
Michal Straka
Keyword(s):  
Hydrogen Bond ◽  
Hydrogen Bonding ◽  
Hydrogen Bonds ◽  
Chemical Shifts ◽  
X Ray ◽  
Structure And Dynamics ◽  
Hydrogen Bonding Interactions ◽  
Overall Stability ◽  
Gold Compounds ◽  
C Nmr

Illusive Au<sup>I/III</sup>···H hydrogen bonds and their effect on structure and dynamics of molecules have been a matter of debate. While a number of X-ray studies reported gold compounds with short Au<sup>I/III</sup>···H contacts, a solid spectroscopic evidence for Au<sup>I/III</sup>···H bonding has been missing. Recently<a></a><a>, Bakar <i>et al.</i></a> (NATURE COMMUNICATIONS 8:576) reported compound with four short Au···H contacts (2.61­–2.66 Å; X-ray determined). Assuming the central cluster be [Au<sub>6</sub>]<sup>2+</sup>and observing the <sup>1</sup>H (<sup>13</sup>C) NMR resonances at relevant H(C) nuclei deshielded with respect to precursor compound, the authors concluded with reservations that <i>“the present Au···H–C interaction is a kind of “hydrogen bond”, where the [Au<sub>6</sub>]<sup>2+</sup>serves as an acceptor”</i>. Here, we show that the Au<sub>6</sub>cluster in their compound bears negative charge and the Au···H contacts lead to a weak (~1 kcal/mol) auride···hydrogen bonding interactions, though unimportant for the overall stability of<b></b>the molecule. Additionally, computational analysis of NMR chemical shifts reveals that the deshielding effects at respective hydrogen nuclei are not directly related to Au···H–C hydrogen bonding .

scholarly journals Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

2014 ◽  
Vol 111 (52) ◽  
pp. 18454-18459 ◽  
Author(s):  
Lu Wang ◽  
Stephen D. Fried ◽  
Steven G. Boxer ◽  
Thomas E. Markland
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Hydrogen Bond Network ◽  
Enzyme Active Site ◽  
Bond Network ◽  
Quantum Delocalization

scholarly journals Hydrogen Bond Coupling in the Ketosteroid Isomerase Active Site

Biochemistry ◽  
2009 ◽  
Vol 48 (29) ◽  
pp. 6932-6939 ◽  
Author(s):  
Paul A. Sigala ◽  
Jose M. M. Caaveiro ◽  
Dagmar Ringe ◽  
Gregory A. Petsko ◽  
Daniel Herschlag
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Ketosteroid Isomerase

scholarly journals Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles

2020 ◽  
Vol 117 (52) ◽  
pp. 33204-33215
Author(s):  
Filip Yabukarski ◽  
Justin T. Biel ◽  
Margaux M. Pinney ◽  
Tzanko Doukov ◽  
Alexander S. Powers ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Enzyme Design ◽  
Reaction Cycle ◽  
New Era ◽  
X Ray ◽  
Enzyme Active Site ◽  
Conformational Ensembles ◽  
X Ray Crystallography ◽  
General Base

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.

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