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 mutants changing residues thought to be involved at the active site were expressed in Escherichia coli, purified to homogeneity by affinity chromatography on a pyridoxamine 5́-phosphate-agarose affinity column, and tested for their kinetic properties and their inactivation by enzyme-activated irreversible inhibitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4% of the total soluble protein), all bound to the affinity column, and there were only small differences in the apparent Km values for L-ornithine providing strong evidence that the mutations did not lead to any gross changes in the protein structure. 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 forms a Schiff base with the pyridoxal 5′-phosphate co-factor. Mutation C70S 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, α-difluoromethylornithine (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 [5-¹⁴C]DFMO was removed from the protein under some denaturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio than the wild type enzyme. These results indicate that inactivation of ODC by DFMO can occur via interaction with either of two separate residues that form essential parts of the active site. This renders it unlikely that resistant mutants will arise from changes in the enzyme structure. In contrast to the results with DFMO, the C360A mutant ODC was completely resistant to inactivation by (R,R)-δ-methyl-a-acetylenicputrescine and was much less sensitive than the wild type enzyme to α-monofluoromethyl dehydromethylornithine, showing that the reactive species formed from these inhibitors either cannot be formed by this mutant or are unable to react with lysine 69. Finally, the well known, extreme reliance of mammalian ODC on the presence of thiol-reducing agents to maintain activity is probably explained by the critical role of cysteine 360, since the C360A mutant was much less sensitive to inactivation by incubation in the absence of dithiothreitol.