Probing the Active Site of the Reconstituted Carnitine Carrier from Rat Liver Mitochondria with Sulfhydryl Reagents. A Cysteine Residue is Localized in or Near the Substrate Binding Site

1995 ◽  
Vol 228 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Cesare Indiveri ◽  
Annamaria Tonazzi ◽  
Nicola Giangregorio ◽  
Ferdinando Palmieri
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Active Site ◽  
Substrate Binding ◽  
Liver Mitochondria ◽  
Cysteine Residue ◽  
Rat Liver Mitochondria ◽  
Sulfhydryl Reagents ◽  
Substrate Binding Site

scholarly journals Binding of [14C]malonyl-CoA to rat liver mitochondria after blocking of the active site of carnitine palmitoyltransferase I. Displacement of low-affinity binding by palmitoyl-CoA

1986 ◽  
Vol 233 (2) ◽  
pp. 589-593 ◽  
Author(s):  
B D Grantham ◽  
V A Zammit
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Active Site ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Malonyl Coa ◽  
High Affinity Site ◽  
Cpt I

The active site of the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria was blocked by the self-catalysed formation of the S-carboxypalmitoyl-CoA ester of (-)-carnitine, followed by washing of the mitochondria. CPT I activity in treated mitochondria was inhibited by 90-95%. Binding of [14C]malonyl-CoA to these mitochondria was not inhibited as compared with that of control mitochondria. When CPT I activity was inhibited, palmitoyl-CoA could markedly displace [14C]malonyl-CoA binding from the low-affinity site for the inhibitor [Zammit, Corstorphine & Gray (1984) Biochem. J. 222, 335-342], but not from the high-affinity site for malonyl-CoA binding. The saturation characteristics of the malonyl-CoA-binding component lost in the presence of palmitoyl-CoA were sigmoidal, and thus suggestive of co-operative binding at this site. It is suggested that the site hitherto considered to be a low-affinity malonyl-CoA-binding site may be effectively a second, allosteric, acyl-CoA-binding site on CPT I under conditions that prevail in vivo, whereas the high-affinity site for malonyl-CoA may be exclusive to the inhibitor. The possibility that the competitive-type interactions of malonyl-CoA and acyl-CoA on CPT I activity could arise from the effects of separate malonyl-CoA and acyl-CoA allosteric sites is considered. The possible significance of the large difference in the capacity of the two sites and their different saturation kinetics is also discussed.

scholarly journals Oxidative phosphorylation. The specific binding of trimethyltin and triethyltin to rat liver mitochondria

1970 ◽  
Vol 118 (1) ◽  
pp. 171-179 ◽  
Author(s):  
W. N. Aldridge ◽  
B. W. Street
Keyword(s):  
Adipose Tissue ◽  
Oxidative Phosphorylation ◽  
Brown Adipose Tissue ◽  
Binding Site ◽  
Rat Liver ◽  
Specific Binding ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Affinity Constants ◽  
Brown Adipose

1. The binding of trimethyltin and triethyltin to rat liver mitochondria was determined and the results were analysed by the method of Scatchard (1949). 2. One binding site (site 1) has the correct characteristics for the site to which trimethyltin and triethyltin are attached when they inhibit oxidative phosphorylation. For each compound the concentration of site 1 is 0.8nmol/mg of protein and the ratios of their affinity constants are the same as the ratio of the concentrations inhibiting oxidative phosphorylation. 3. Binding site 1 is present in a fraction derived from mitochondria containing only 15% of the original protein. In this preparation ultrasonication rapidly destroyed site 1. 4. Dimethyltin and diethyltin do not prevent binding of triethyltin to rat liver mitochondria, whereas triethyl-lead does. 5. Trimethyltin and triethyltin bind to mitochondria from brown adipose tissue and the results indicate a binding site 1 similar to that in rat liver mitochondria. 6. The advantages and limitations of this approach to the study of inhibitors are discussed.

scholarly journals Studies on the nature of the high-affinity trialkyltin binding site of rat liver mitochondria

1982 ◽  
Vol 202 (1) ◽  
pp. 163-169 ◽  
Author(s):  
A P Dawson ◽  
B G Farrow ◽  
M J Selwyn
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Affinity Binding ◽  
Mitochondrial Atpase ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Triphenyltin Chloride

1. The proteolipid fraction isolated from rat liver mitochondria pretreated with [3H]triphenyltin chloride is enriched in triphenyltin compared with the original mitochondria. 2. Part of this [3H]triphenyltin is eluted with a protein of Mr 5000-6000 on Sephadex LH20 chromatography. 2. Mössbauer spectra of the proteolipid fraction treated with 119Sn-enriched triethyltin chloride show a doublet which corresponds closely with that assigned previously [Farrow & Dawson (1978) Eur. J. Biochem. 86. 85-95] to the absorption of triethyltin bound to the high-affinity binding site of the mitochondrial ATPase.

Crystal structures of substrate-bound chitinase from the psychrophilic bacteriumMoritella marinaand its structure in solution

2014 ◽  
Vol 70 (3) ◽  
pp. 676-684 ◽  
Author(s):  
Piotr H. Malecki ◽  
Constantinos E. Vorgias ◽  
Maxim V. Petoukhov ◽  
Dmitri I. Svergun ◽  
Wojciech Rypniewski
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Domain Structure ◽  
Active Site ◽  
Substrate Binding ◽  
Glycoside Hydrolases ◽  
Substrate Binding Site ◽  
Domain Structures ◽  
Structure In Solution

The four-domain structure of chitinase 60 fromMoritella marina(MmChi60) is outstanding in its complexity. Many glycoside hydrolases, such as chitinases and cellulases, have multi-domain structures, but only a few have been solved. The flexibility of the hinge regions between the domains apparently makes these proteins difficult to crystallize. The analysis of an active-site mutant ofMmChi60 in an unliganded form and in complex with the substrates NAG4and NAG5revealed significant differences in the substrate-binding site compared with the previously determined complexes of most studied chitinases. A SAXS experiment demonstrated that in addition to the elongated state found in the crystal, the protein can adapt other conformations in solution ranging from fully extended to compact.

scholarly journals Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

2011 ◽  
Vol 77 (16) ◽  
pp. 5730-5738 ◽  
Author(s):  
Hanna M. Dudek ◽  
Gonzalo de Gonzalo ◽  
Daniel E. Torres Pazmiño ◽  
Piotr Stępniak ◽  
Lucjan S. Wyrwicz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Active Site ◽  
Structural Model ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Thermobifida Fusca ◽  
Content Type ◽  
Substrate Binding Site

ABSTRACTBaeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) fromThermobifida fuscais the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

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