KINETICS OF INHIBITION OF RABBIT RETICULOCYTE PEPTIDYLTRANSFERASE BY ANISOMYCIN AND SPARSOMYCIN

A detailed kinetic study was carried out on the inhibitory mechanisms of two eukaryotic peptidyltransferase drugs (I), anisomycin and sparso mycin. In an in vitro system from rabbit reticulocytes, AcPhe-puromyci n is produced in a pseudo-first-order reaction from the preformed AcPh e-tRNA/poly(U)/80S ribosome complex (complex C) and excess puromycin ( S). This reaction is inhibited by anisomycin and sparsomycin through d ifferent mechanisms. Anisomycin acts as a mixed noncompetitive inhibit or. The product, AcPhe-puromycin, is derived only from C according to the puromycin reaction. On the other hand, sparsomycin reacts with com plex C in a two-step reaction, [GRAPHICS] An initial rapid binding of the drug produces the encounter complex Cl. During this step and befor e conversion of Cl to C(star)l, sparsomycin behaves as a competitive i nhibitor. The rapidly produced Cl is isomerized slowly to a conformati onally altered species (CI)-I-star in which I is bound more tightly. T he rate constants of this step are k(6) = 2.1 min(-1) and k(7) = 0.095 min(-1). Moreover, the low value of the association rate constant k(7 )/K-i' (2 x 10(5) M-1 sec(-1)), provides insight into the rates of pos sible conformational changes occurring during protein synthesis and su pports the proposal that sparsomycin is the first example of a slow-bi nding inhibitor of eukaryotic peptidyltransferase. When complex C is p reincubated with concentrations of sparsomycin of >8 K-i and then reac ts with a mixture of puromycin and sparsomycin, the inhibition becomes linear mixed noncompetitive and involves C(star)l instead of Cl. Duri ng this phase, AcPhe-puromycin is produced from a new, modified riboso mal complex with a lower catalytic rate constant. Thus, sparsomycin al so acts as a modifier of eukaryotic peptidyltransferase activity.