Role of the Heme Active Site and Protein Environment in Structure, Spectra, and Function of the Cytochrome P450s

2000 ◽  
Vol 100 (2) ◽  
pp. 407-420 ◽  
Author(s):  
Gilda H. Loew ◽  
Danni L. Harris
Keyword(s):  
Active Site ◽  
Cytochrome P450s ◽  
And Function ◽  
Protein Environment

Characterization of anEscherichia coliSulfite Oxidase Homologue Reveals the Role of a Conserved Active Site Cysteine in Assembly and Function†

Biochemistry ◽  
2005 ◽  
Vol 44 (30) ◽  
pp. 10339-10348 ◽  
Author(s):  
Stephen J. Brokx ◽  
Richard A. Rothery ◽  
Guijin Zhang ◽  
Derek P. Ng ◽  
Joel H. Weiner
Keyword(s):  
Active Site ◽  
Active Site Cysteine ◽  
And Function

scholarly journals The role of endothelium in factor Xa regulation: the effect of plasma proteinase inhibitors and hirudin

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1321-1328 ◽  
Author(s):  
RC Friedberg ◽  
PO Hagen ◽  
SV Pizzo
Keyword(s):  
Active Site ◽  
Proteinase Inhibitor ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Proteinase Inhibitors ◽  
Factor Xa ◽  
Proteolytic Degradation ◽  
Chromogenic Substrates ◽  
And Function

Abstract The role of endothelium in the inhibition of human factor Xa was studied in a plasma environment. Human factor Xa can bind to and function on bovine aortic endothelium in a manner similar to that of bovine factor Xa. Approximately 70% of the bound factor Xa is subject to inhibition by plasma proteinase inhibitors, and the remaining 30% is irreversibly bound as part of a 125 Kd membrane-associated complex not subject to proteolytic degradation. The proportion reversibly bound and its rate of release do not alter with changes in calcium, citrate, heparin, or active proteinase inhibitor concentrations. The principal plasma proteinase inhibitor of human factor Xa was antithrombin III, which accounted for 60% to 65% of factor Xa released from endothelium, with alpha 1-proteinase inhibitor inactivating 20% to 25% and alpha 2- macroglobulin approximately 15%. All of the reversibly bound factor Xa was identified in complex with one of these three proteinase inhibitors. The thrombin active-site inhibitor hirudin was found to markedly accelerate the displacement of reversibly bound factor Xa from the endothelium and to associate specifically with factor Xa without a loss of activity toward chromogenic substrates, perhaps accounting for a novel mechanism of anticoagulation.

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.

Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site

2005 ◽  
Vol 33 (5) ◽  
pp. 1189-1196 ◽  
Author(s):  
W.D. Nes
Keyword(s):  
Active Site ◽  
Functional Divergence ◽  
Product Distribution ◽  
Site Directed Mutagenesis ◽  
Site Model ◽  
Bifunctional Enzymes ◽  
And Function ◽  
The Relationship ◽  
Sterol Methyltransferase

Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Δ24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.

scholarly journals Dioxygen Binding in the Active Site of Histone Demethylase JMJD2A and the Role of the Protein Environment

2015 ◽  
Vol 21 (52) ◽  
pp. 18869-18869
Author(s):  
Wilian A. Cortopassi ◽  
Robert Simion ◽  
Charles E. Honsby ◽  
Tanos C. C. França ◽  
Robert S. Paton
Keyword(s):  
Active Site ◽  
Histone Demethylase ◽  
Protein Environment

Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

2013 ◽  
Vol 1834 (3) ◽  
pp. 688-696 ◽  
Author(s):  
Jeanette E. Stok ◽  
Sean Yamada ◽  
Anthony J. Farlow ◽  
Kate E. Slessor ◽  
James J. De Voss
Keyword(s):  
Active Site ◽  
Cytochrome P450s

scholarly journals Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

2015 ◽  
Vol 60 (1) ◽  
pp. 356-360 ◽  
Author(s):  
Asad U. Khan ◽  
M. Tabish Rehman
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Structure And Function ◽  
Clinical Strain ◽  
New Delhi ◽  
Active Site Residue ◽  
Active Site Residues ◽  
Content Type ◽  
And Function

ABSTRACTNew Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members ofEnterobacteriaceaeas a defense mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We clonedblaNDM-1from anEnterobacter cloacaeclinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (theKmwas reduced, while thekcatwas similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ΔGDoandTmof Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.

scholarly journals The role of endothelium in factor Xa regulation: the effect of plasma proteinase inhibitors and hirudin

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1321-1328
Author(s):  
RC Friedberg ◽  
PO Hagen ◽  
SV Pizzo
Keyword(s):  
Active Site ◽  
Proteinase Inhibitor ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Proteinase Inhibitors ◽  
Factor Xa ◽  
Proteolytic Degradation ◽  
Chromogenic Substrates ◽  
And Function

The role of endothelium in the inhibition of human factor Xa was studied in a plasma environment. Human factor Xa can bind to and function on bovine aortic endothelium in a manner similar to that of bovine factor Xa. Approximately 70% of the bound factor Xa is subject to inhibition by plasma proteinase inhibitors, and the remaining 30% is irreversibly bound as part of a 125 Kd membrane-associated complex not subject to proteolytic degradation. The proportion reversibly bound and its rate of release do not alter with changes in calcium, citrate, heparin, or active proteinase inhibitor concentrations. The principal plasma proteinase inhibitor of human factor Xa was antithrombin III, which accounted for 60% to 65% of factor Xa released from endothelium, with alpha 1-proteinase inhibitor inactivating 20% to 25% and alpha 2- macroglobulin approximately 15%. All of the reversibly bound factor Xa was identified in complex with one of these three proteinase inhibitors. The thrombin active-site inhibitor hirudin was found to markedly accelerate the displacement of reversibly bound factor Xa from the endothelium and to associate specifically with factor Xa without a loss of activity toward chromogenic substrates, perhaps accounting for a novel mechanism of anticoagulation.

Cover Picture: Dioxygen Binding in the Active Site of Histone Demethylase JMJD2A and the Role of the Protein Environment (Chem. Eur. J. 52/2015)

2015 ◽  
Vol 21 (52) ◽  
pp. 18865-18865
Author(s):  
Wilian A. Cortopassi ◽  
Robert Simion ◽  
Charles E. Honsby ◽  
Tanos C. C. França ◽  
Robert S. Paton
Keyword(s):  
Active Site ◽  
Histone Demethylase ◽  
Protein Environment
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