A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I.
The requirements for binding at the N-acetyl-L-glutamate binding site of carbamoyl phosphate synthetase I were studied by the displacement of the activator from the central enzyme complex by analogs. Two carboxyls are essential and the acetamido group, if linked to the alpha-carbon, enhances binding 5000-fold. The subsite for the delta-carboxyl is mobile with respect to that for the alpha-carboxyl. Mixtures of complementary fragment of acetylglutamate do not bind, indicating a strong 'chelate' effect. Substituents revealed the existence of steric constraints around the delta-carboxyl, the alpha and gamma-carbons, and the whole of the acetamido group. However, phenyl substituents at the beta-carbon did not hamper binding, indicating that substituents at the beta-carbon face the solution. This is consistent with binding of acetylglutamate as the minimum-energy conformer. All analogs binding with high affinity are activators. Some analogs that bind poorly are competitive inhibitors. They appear to bind preferentially to a low-affinity conformation adopted by the site when the products dissociate and the substrates bind. The acetamido group plays no role in the binding of the inhibitors but it is crucial for the binding of the activators, and the high- and low-affinity conformations of the site differ markedly in structural selectivity.
Britton
H G
HG
Department of Physiology, St. Mary's Hospital Medical School, London, England.
García-España
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A
Goya
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P
Rozas
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Rubio
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D000081:Acetamides / Q000378:metabolism; D000107:Acetylation; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D011487:Protein Conformation; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000081:Acetamides; D002264:Carboxylic Acids; D005971:Glutamates; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid