Potentiometric and Further Kinetic Characterization of the Flavin-Binding Domain ofSaccharomycescerevisiaeFlavocytochromeb2. Inhibition by Anions Binding in the Active Site†

Biochemistry ◽  
2007 ◽  
Vol 46 (15) ◽  
pp. 4661-4670 ◽  
Author(s):  
Narimantas Čénas ◽  
K. H. Diêp Lê ◽  
Micheline Terrier ◽  
Florence Lederer
Keyword(s):  
Active Site ◽  
Binding Domain ◽  
Kinetic Characterization ◽  
Flavin Binding

scholarly journals Structure and Mechanism of Human UDP-glucose 6-Dehydrogenase

2011 ◽  
Vol 286 (27) ◽  
pp. 23877-23887 ◽  
Author(s):  
Sigrid Egger ◽  
Apirat Chaikuad ◽  
Kathryn L. Kavanagh ◽  
Udo Oppermann ◽  
Bernd Nidetzky
Keyword(s):  
Active Site ◽  
Quaternary Structure ◽  
Binding Domain ◽  
Side Chain ◽  
Reaction Conditions ◽  
Epithelial Tumor ◽  
Coenzyme Binding ◽  
Ternary Complex Formation ◽  
The Matrix

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD+-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD+. The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD+ binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr14, Glu161, and Glu165 participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys276 functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys276 in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD+ binding domain.

scholarly journals Thermodynamic and Kinetic Characterization of the Interaction between the Ras Binding Domain of AF6 and Members of the Ras Subfamily

1999 ◽  
Vol 274 (19) ◽  
pp. 13556-13562 ◽  
Author(s):  
Thomas Linnemann ◽  
Matthias Geyer ◽  
Birgit K. Jaitner ◽  
Christoph Block ◽  
Hans Robert Kalbitzer ◽  
...  
Keyword(s):  
Binding Domain ◽  
Kinetic Characterization

Spectroscopic and kinetic characterization of active site mutants of Desulfovibrio fructosovorans Ni-Fe hydrogenase

2003 ◽  
Vol 8 (1) ◽  
pp. 129-134 ◽  
Author(s):  
Antonio L. De Lacey ◽  
Victor M. Fernandez ◽  
Marc Rousset ◽  
Christine Cavazza ◽  
Claude E. Hatchikian
Keyword(s):  
Active Site ◽  
Kinetic Characterization ◽  
Active Site Mutants

scholarly journals Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli

1995 ◽  
Vol 309 (2) ◽  
pp. 601-605 ◽  
Author(s):  
A Balme ◽  
C E Brunt ◽  
R L Pallister ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Escherichia Coli ◽  
Binding Domain ◽  
Flavin Mononucleotide ◽  
Exponential Process ◽  
Flavocytochrome B2 ◽  
Terminal Domain ◽  
Isolation And Characterization ◽  
Flavin Binding ◽  
Single Exponential

Flavocytochrome b2 consists of two distinct domains. The N-terminal domain contains protohaem IX and the larger, C-terminal domain contains flavin mononucleotide (FMN). We describe here the isolation of the flavin-binding domain expressed in Escherichia coli independent of the cytochrome domain. The isolated domain is an efficient lactate dehydrogenase with ferricyanide as electron acceptor but reduces cytochrome c, the physiological oxidant for flavocytochrome b2, extremely poorly; electron transfer from the flavin-binding domain to the separately expressed cytochrome domain is undetectable. FMN reduction by lactate occurs as a single exponential process in the isolated flavin-binding domain, in contrast to the biphasic kinetics observed with native flavocytochrome b2.

Structural and Kinetic Characterization of Active-Site Histidine as a Proton Shuttle in Catalysis by Human Carbonic Anhydrase II†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (4) ◽  
pp. 1097-1105 ◽  
Author(s):  
Zoë Fisher ◽  
Jose A. Hernandez Prada ◽  
Chingkuang Tu ◽  
David Duda ◽  
Craig Yoshioka ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Active Site ◽  
Carbonic Anhydrase Ii ◽  
Kinetic Characterization ◽  
Human Carbonic Anhydrase ◽  
Active Site Histidine

Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin‐dependent N‐oxygenase

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Katherine Huynh ◽  
Taylor Hardin ◽  
Lucero Guzman ◽  
Nadia Castillejo ◽  
Bruce Palfey ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Kinetic Characterization ◽  
Flavin Binding
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