Lysine-69 plays a key role in catalysis by ornithine decarboxylase throughacceleration of the Schiff base formation, decarboxylation, and product release steps

Ornithine decarboxylase (ODC) is a pyridoxal-5'-phosphate-dependent (PLP) e nzyme that catalyzes the biosynthesis of the polyamine putrescine. Similar to other PLP-dependent enzymes, an active site Lys residue forms a Schiff b ase with PLP in the absence of substrate. The mechanistic role of this resi due (Lys-69) in catalysis by Trypanosoma brucei ODC has been studied by ana lysis of the mutant enzymes, in which Lys-69 has been replaced by Arg (K69R ODC) and Ala (K69A ODC).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 ac tive site through a Schiff base with PLP. In contrast, on the basis of an a bsorption spectrum of K69R ODC, PLP is likely to be bound to this mutant en zyme in the aldehyde form. Pre-steady-state kinetic analysis of the reactio n 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 by10(4)-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 10(3)-fold. Pro duct release remains as the dominant rate-limiting step in the reaction (th e 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 sugges ts that Lys-69 may play a role in the proper positioning of the cl-carboxyl ate for efficient decarboxylation. K69R ODC binds diamines and amino acids with higher affinity than the wild-type enzyme; however, Lys-69 does not me diate substrate specificity. Wild-type and K69R ODC have similar ligand spe cificity preferring to bind putrescine over longer and shorter diamines. Ki netic analysis of the binding of a series of diamines and amino acids to K6 9R ODC suggests that noncovalent interactions in the active site of K69R OD C promote selective ligand binding during Schiff base formation.