RT Journal Article SR Electronic T1 Kinetics of Inhibition of Rabbit Reticulocyte Peptidyltransferase by Anisomycin and Sparsomycin JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1089 OP 1096 VO 53 IS 6 A1 Margarita Ioannou A1 Charalambos Coutsogeorgopoulos A1 Dennis Synetos YR 1998 UL http://molpharm.aspetjournals.org/content/53/6/1089.abstract AB A detailed kinetic study was carried out on the inhibitory mechanisms of two eukaryotic peptidyltransferase drugs (I), anisomycin and sparsomycin. In an in vitro system from rabbit reticulocytes, AcPhe-puromycin is produced in a pseudo-first-order reaction from the preformed AcPhe-tRNA/poly(U)/80S ribosome complex (complex C) and excess puromycin (S). This reaction is inhibited by anisomycin and sparsomycin through different mechanisms. Anisomycin acts as a mixed noncompetitive inhibitor. The product, AcPhe-puromycin, is derived only from C according to the puromycin reaction. On the other hand, sparsomycin reacts with complex C in a two-step reaction,    C+I  Ki ⇌ CI k6⇌k7 C∗I   An initial rapid binding of the drug produces the encounter complex CI. During this step and before conversion of CI to C*I, sparsomycin behaves as a competitive inhibitor. The rapidly produced CI is isomerized slowly to a conformationally altered species C*I in which I is bound more tightly. The rate constants of this step arek6 = 2.1 min−1 andk7 = 0.095 min−1. Moreover, the low value of the association rate constantk7/Ki ′ (2 × 105m−1sec−1), provides insight into the rates of possible conformational changes occurring during protein synthesis and supports the proposal that sparsomycin is the first example of a slow-binding inhibitor of eukaryotic peptidyltransferase. When complex C is preincubated with concentrations of sparsomycin of >8Ki and then reacts with a mixture of puromycin and sparsomycin, the inhibition becomes linear mixed noncompetitive and involves C*I instead of CI. During this phase, AcPhe-puromycin is produced from a new, modified ribosomal complex with a lower catalytic rate constant. Thus, sparsomycin also acts as a modifier of eukaryotic peptidyltransferase activity. The American Society for Pharmacology and Experimental Therapeutics