The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible. These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)-site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs). This kobs varies with the concentration (of puromycin; the maximal value (k3) of the kobsr at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin. k 3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat).If we assume that the puromycin-reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P-site binding of the donor substrates AcPhetRNA or fMet-tRNA can be the same and close to 100Y0, while there is a tenfold increase in the reactivity of the donor ( k 3 = 0.8 min-versus 8.3 min-I). On the other hand there are conditions, u.nder which the proportion of P-site binding increases from 30% to 100% while k 3 remains low and equal to 0.8 min-'. Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se. The fact that the donor substrate may exist in various states of reactivity in this cell-free system raises the possibility thal: the rate of peptide bond formation may not be uniform during protein synthesis.The puromycin reaction is the reaction in which a peptide bond is formed between peptidyl-tRNA bound to ribosomes and the antibiotic puromycin. It has been used to determine the ribosomal site location of bound peptidyl-tRNA on an operational basis [l -31. Thus, if peptidyl-tRNA reacts with puromycin, its location is believed to be the P, or peptidyl, site of the ribosome. If, on the other hand, peptidyl-tRNA does not react with puromycin, then it is located at other site(s) of the ribosome 11, 4-61.The puromycin reaction has been employed by a large number of workers in various forms [7 -101. In these studies the final degree of the puromycin reaction has been estimated. This parameter, usually called the extent of the reaction [lo], denotes the proportion of P-sitc-bound donor which is reactive to puromycin and is often referred to as reactivity towards puromycin [7,9, 101. We have already used the puromycin reaction in order to define the activity of peptidyltransferase which catalyzes this reaction [l I]. Here we define the reactivity of the P-site-bound donor substrate toward puromycin based on the analysis of kinetic information and we differentiate this information from that given by other workers. We determine the reactivity of the donor via a rate constant (...