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N5-(l-1-carboxyethyl)-l-ornithine synthase: Physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues

Published online by Cambridge University Press:  01 October 1999

DAN L. SACKETT
Affiliation:
Laboratory of Integrative and Medical Biophysics, NICHD, National Institutes of Health, Bethesda, Maryland 20892
SERGEI B. RUVINOV
Affiliation:
Section on Protein Chemistry, Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
JOHN THOMPSON
Affiliation:
Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892
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Abstract

N5-(l-1-carboxyethyl)-l-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr ≅ 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the α,β family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of ∼8.5, 11 have pKa ∼ 10.75, and the remaining six titrate with pKa ∼ 11.3. The two residues with pKa ∼ 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced 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 proximity to the sequence GSGNVA, that constitutes the βαβ 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 stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.

Type
Research Article
Copyright
© 1999 The Protein Society

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