Three-Point Binding Model课件.pptVIP

Three-Point Binding Model First proposed by Ogsten (1948) to explain biological enantioselection/enantiospecificity Serves as a model for chromatographic chiral stationary phases Enantioselection by an Enzyme Three-Point Binding Model - Enantiospecificity Only one enantiomer binds to enzyme is involved in reaction ? we get enantiospecificity (substrate biomolecule are chiral) To do this efficiently, we need a large biomolecule to align three binding sites to give high specificity One problem with model: Model is a static representation → “lock key” Binding The cost of binding: Km (Michaelis constant): small value indicates high affinity for substrate ? Kbinding ( ~ 1/Km) Strong binding → K 1 ΔG= -RT ln K ΔG must be –ve ΔGbinding = ΔHbinding- TΔSbinding For 2 molecules in, 1 out: ΔS is –ve ? (-TΔS) term is +ve Entropy disfavors binding of substrate to enzyme To get good binding, need –ve ΔH (i.e. bond formation) Each non-covalent interaction is small (H-bond ~ 5 kcal/mol), but still gives a –ve ΔH Enzymes use many FG’s to sum up many weak non-covalent interactions (i.e. 3 points) Back to tyrosyl-tRNA synthase: Tyrosyl-tRNA synthase Use binding to orient CO2- nucleophile adjacent to P? specifically as electrophile → specificity Many non-covalent interactions overcome entropy of binding: H-bonds Tyrosyl-tRNA Synthase.tyr-adenylate We have examined the crystal structure of tyrosyl-tRNA synthase (Tyr ATP bound) Key contacts 3 point binding model for (S)-tyrosine We inferred geometry of bound ATP prior to reaction (i.e. ATP is no longer bound to enzyme) Step 1: CO2- attacks PO42- (?) giving pentacoordinate P (trigonal bipyramidal) intermediate Step 2: Diphosphate must leave Cannot “see” this step ? PPi has already left the enzyme site in the crystal structure However, can use model building to include P? P? of ATP: Tests of Mechanism Site-directed mutagenesis Replace Gln195 with Gly ? (Gln195Gly) Rate slows by 1000 fold ΔΔG? ~ 4