scholarly journals Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

ACS Catalysis ◽  
2015 ◽  
Vol 5 (2) ◽  
pp. 892-899 ◽  
Author(s):  
Ashley B. Daugherty ◽  
John R. Horton ◽  
Xiaodong Cheng ◽  
Stefan Lutz
Keyword(s):  
Active Site ◽  
Circular Permutation ◽  
Old Yellow Enzyme ◽  
Functional Consequences

scholarly journals Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets

1986 ◽  
Vol 239 (2) ◽  
pp. 301-310 ◽  
Author(s):  
W D Sweet ◽  
F Schroeder
Keyword(s):  
Plasma Membrane ◽  
Active Site ◽  
Plasma Membranes ◽  
Local Anaesthetics ◽  
Cationic Drugs ◽  
Cell Plasma ◽  
Composition And Structure ◽  
Highly Sensitive ◽  
Functional Consequences ◽  
Anionic Drugs

The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5′-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.

scholarly journals Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

2022 ◽  
Author(s):  
Andrea Piserchio ◽  
Eta A Isiroho ◽  
Kimberly Long ◽  
Amanda L Bohanon ◽  
Eric A Kumar ◽  
...  
Keyword(s):  
Active Site ◽  
Protein Quality ◽  
Regulatory Element ◽  
Structural Basis ◽  
Calmodulin Binding ◽  
Intimate Association ◽  
Biochemical Studies ◽  
Functional Consequences ◽  
Heterodimeric Complex ◽  
Elongation Phase

Translation is a highly energy consumptive process tightly regulated for optimal protein quality and adaptation to energy and nutrient availability. A key facilitator of this process is the α-kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process mechanistically distinct from other calmodulin-dependent kinases. Here we resolve the atomic details of this mechanism through a 2.3 Å crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a spine that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

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.

scholarly journals On the Active Site of Old Yellow Enzyme

1998 ◽  
Vol 273 (49) ◽  
pp. 32753-32762 ◽  
Author(s):  
Bette Jo Brown ◽  
Zhan Deng ◽  
P. Andrew Karplus ◽  
Vincent Massey
Keyword(s):  
Active Site ◽  
Old Yellow Enzyme
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