Dataset Information

Unnatural amino acid substitution as a probe of the allosteric coupling pathway in a mycobacterial Cu(I) sensor.

ABSTRACT: The Cu(I) sensor Mycobacterium tuberculosis CsoR is a founding member of a new metalloregulatory protein family. Here we show that two "atom" substitutions of the Nepsilon2 face of a Cu(I) coordinating histidine-61 allosterically uncouple Cu(I) and DNA binding, with no effect on Cu(I) binding affinity and coordination structure. A model analogous to the allosteric switch mechanism in Staphylococcus aureus CzrA, a zinc sensor protein with a completely different fold, is proposed.

SUBMITTER: Ma Z

PROVIDER: S-EPMC2797707 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Unnatural amino acid substitution as a probe of the allosteric coupling pathway in a mycobacterial Cu(I) sensor.

Ma Zhen Z Cowart Darin M DM Ward Brian P BP Arnold Randy J RJ DiMarchi Richard D RD Zhang Limei L George Graham N GN Scott Robert A RA Giedroc David P DP

Journal of the American Chemical Society 20091201 50

The Cu(I) sensor Mycobacterium tuberculosis CsoR is a founding member of a new metalloregulatory protein family. Here we show that two "atom" substitutions of the Nepsilon2 face of a Cu(I) coordinating histidine-61 allosterically uncouple Cu(I) and DNA binding, with no effect on Cu(I) binding affinity and coordination structure. A model analogous to the allosteric switch mechanism in Staphylococcus aureus CzrA, a zinc sensor protein with a completely different fold, is proposed. ...[more]

PMID: 19928961

Similar Datasets

Project description:Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via "short, strong" or "low-barrier" hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater "sensitivity") to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine with fluorotyrosines (F-Tyr's) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic environment that greatly enhances the energetic response to charge accumulation, consistent with prior experimental results.

Dataset Information

Unnatural amino acid substitution as a probe of the allosteric coupling pathway in a mycobacterial Cu(I) sensor.

Publications

Unnatural amino acid substitution as a probe of the allosteric coupling pathway in a mycobacterial Cu(I) sensor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets