Ornithine decarboxylase (ODC) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the biosynthesis of the polyamine putrescine. Similar to other PLP-dependent enzymes, an active site Lys residue forms a Schiff base with PLP in the absence of substrate. The mechanistic role of this residue (Lys-69) in catalysis by Trypanosoma brucei ODC has been studied by analysis of the mutant enzymes, in which Lys-69 has been replaced by Arg (K69R ODC) and Ala (K69A ODC). Analysis of K69A ODC demonstrated that the enzyme copurified with amines (e.g. putrescine) that were tightly bound to the active site through a Schiff base with PLP. In contrast, on the basis of an absorption spectrum of K69R ODC, PLP is likely to be bound to this mutant enzyme in the aldehyde form. Pre-steady-state kinetic analysis of the reaction of K69R ODC with L-Orn and putrescine demonstrated that the rates of both the product release (k(off.Put)= 0.0041 s-1) and the decarboxylation (k(decarb) = 0.016 s-1) steps were decreased by 104-fold in comparison to wild-type ODC. Further, the rates of Schiff base formation between K69R ODC and either substrate or product have decreased by at least 103-fold. Product release remains as the dominant rate-limiting step in the reaction (the steady-state parameters for K69R ODC are k(cat) = 0.0031 s-1 and K(m)= 0.18 mM). The effect of mutating Lys-69 on the decarboxylation step suggests that Lys-69 may play a role in the proper positioning of the α-carboxylate for efficient decarboxylation. K69R ODC binds diamines and amino acids with higher affinity than the wild-type enzyme; however, Lys-69 does not mediate substrate specificity. Wild-type and K69R ODC have similar ligand specificity preferring to bind putrescine over longer and shorter diamines. Kinetic analysis of the binding of a series of diamines and amino acids to K69R ODC suggests that noncovalent interactions in the active site of K69R ODC promote selective ligand binding during Schiff base formation.
|Original language||English (US)|
|Number of pages||13|
|State||Published - Sep 7 1999|
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