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, [REACTION; SEE TEXT] 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 are k6 = 2.1 min-1 and k7 = 0.095 min-1. Moreover, the low value of the association rate constant k7/Ki' (2 x 10(5) M-1 sec-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 >8 Ki 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.