PMID:2958087
| Citation |
Lau, FT, Fersht, AR, Hellinga, HW and Evans, PR (1987) Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase. Biochemistry 26:4143-8 |
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| Abstract |
A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55. The dissociation constant of PEP from the T state is unaffected by the mutations. Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding. A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP. |
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| Keywords |
Binding Sites/drug effects; Enzyme Activation/drug effects; Escherichia coli/enzymology; Genetic Engineering; Glycine/genetics; Guanosine Diphosphate/pharmacokinetics; Guanosine Diphosphate/pharmacology; Kinetics; Mutation; Phosphoenolpyruvate/pharmacokinetics; Phosphoenolpyruvate/pharmacology; Phosphofructokinase-1/antagonists & inhibitors; Phosphofructokinase-1/genetics; Tyrosine/genetics |
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