N-5-(L-1-carboxyethyl)-L-ornithine synthase: Physical and spectral characterization of the enzyme and its unusual low pK(a) fluorescent tyrosine residues

N-5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lac tococcus lactis has been cloned, ex pressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (M-r congruent to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers, The far-ultraviolet circular dichroism spectrum shows that the fo lding pattern of the enzyme is typical of the alpha,beta family of proteins . CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, an d the fluorescence spectrum of the protein shows emission from both Trp and Sr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of th e native enzyme is about 0.5. All 19 Tyr residues are titratable and, of th ese, two exhibit the uncommonly low pK(a) of similar to 8.5, 11 have pK(a) similar to 10.75, and the remaining six titrate with pK(a) 11.3. The two re sidues with pK(a) similar to 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicatin g Tyr-Tyr energy transfer. In the presence of NADPH, Sr fluorescence is red uced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in p roximity to the sequence GSGNVA, that constitutes the beta alpha beta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stabil ity is maintained. Possible roles in catalysis for the chymotrypsin sensiti ve loop and for the low pK(a) Tyr residues are discussed.