Kinetic study of irreversible inhibition of an enzyme consumed in the reaction it catalyses. Application to the inhibition of the puromycin reaction by spiramycin and hydroxylamine.
A systematic procedure for the kinetic study of irreversible inhibition when the enzyme is consumed in the reaction which it catalyses, has been developed and analysed. Whereas in most reactions the enzymes are regenerated after each catalytic event and serve as reusable transacting effectors, in the consumed enzymes each catalytic center participates only once and there is no enzyme turnover. A systematic kinetic analysis of irreversible inhibition of these enzyme reactions is presented. Based on the algebraic criteria proposed in this work, it should be possible to evaluate either the mechanism of inhibition (complexing or non-complexing), or the type of inhibition (competitive, non-competitive, uncompetitive, mixed non-competitive). In addition, all kinetic constants involved in each case could be calculated. An experimental application of this analysis is also presented, concerning peptide bond formation in vitro. Using the puromycin reaction, which is a model reaction for the study of peptide bond formation in vitro and which follows the same kinetic law as the enzymes under study, we have found that: (i) the antibiotic spiramycin inhibits the puromycin reaction as a competitive irreversible inhibitor in a one step mechanism with an association rate constant equal to 1.3 x 10(4) M-1 s-1 and, (ii) hydroxylamine inhibits the same reaction as an irreversible non-competitive inhibitor also in a one step mechanism with a rate constant equal to 1.6 x 10(-3) M-1 s-1.
Dinos
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Laboratory of Biochemistry, School of Medicine, University of Patras, Greece.
Coutsogeorgopoulos
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D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli; D019811:Hydroxylamine / Q000494:pharmacology*; D008956:Models, Chemical; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors; D011500:Protein Synthesis Inhibitors; D011691:Puromycin / Q000037:antagonists & inhibitors*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D015572:Spiramycin / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D012346:RNA, Transfer, Amino Acyl; D019811:Hydroxylamine; D011691:Puromycin; D015572:Spiramycin; D000217:Acyltransferases; D010458:Peptidyl Transferases