EFFECT OF MUTATIONS AT ACTIVE-SITE RESIDUES ON THE ACTIVITY OF ORNITHINE DECARBOXYLASE AND ITS INHIBITION BY ACTIVE-SITE-DIRECTED IRREVERSIBLE INHIBITORS

Mouse ornithine decarboxylase (ODC) and mutant changing residues thoug ht to be involved at the active site were expressed in Escherichia col i, purified to homogeneity by affinity chromatography on a pyridoxamin e 5'-phosphate-agarose affinity column, and tested for their kinetic p roperties and their inactivation by enzyme-activated irreversible inhi bitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4% of the total soluble protein), all boun d to the affinity column, and there were only small differences in the apparent K(m) values for L-ornithine providing strong evidence that t he mutations did not lead to any gross changes in the protein structur e. The mutation K69A led to a change in the spectrum of the enzyme and a 550-fold decrease in the k(cat)/K(m) (specificity constant) value. These results are consistent with lysine 69 being the residue that for ms a Schiff base with the pyridoxal 5'-phosphate co-factor. Mutation C 70S did not greatly affect the activity despite its proximity to this lysine but increased the K(m) about 2-fold. In contrast, the mutation C360A greatly reduced the specificity constant (by 26-fold) despite a 2-fold decrease in the K(m), suggesting that this cysteine residue is critically involved at the active site. Although cysteine 360 is known to be the major site of binding of the inhibitor, alpha-difluoromethy lornithine (DFMO), the C360A mutant was still sensitive to inhibition by this drug. However, the kinetics of inactivation were altered, the partition ratio was 10 times greater, and the labeled adduct formed by reaction with [5-C-14]DFMO was removed from the protein under some de naturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio th an the wild type enzyme. These results indicate that inactivation of O DC by DFMO can occur via interaction with either of two separate resid ues that form essential parts of the active site. This renders it unli kely that resistant mutants will arise from changes in the enzyme stru cture. In contrast to the results with DFMO, the C360A mutant ODC was completely resistant to inactivation by (R,R)-delta-methyl-alpha-acety lenicputrescine and was much less sensitive than the wild type enzyme to alpha-monofluoromethyldehydromethylornithine, showing that the reac tive species formed from these inhibitors either cannot be formed by t his mutant or are unable to react with lysine 69. Finally, the well kn own, extreme reliance of mammalian ODC on the presence of thiol-reduci ng agents to maintain activity is probably explained by the critical r ole of cysteine 360, since the C360A mutant was much less sensitive to inactivation by incubation in the absence of dithiothreitol.