J. Biol. Chem., Vol. 259, Issue 1, 249-254, Jan, 1984

Hydroxyproline 2-epimerase of Pseudomonas. Subunit structure and active site studies

SG Ramaswamy

Hydroxyproline 2-epimerase of Pseudomonas putida was purified to homogeneity by an improved procedure. The native enzyme consists of two probably identical subunits. Alkylation of the active site with labeled reagents resulted in the loss of 80-85% of the activity but the incorporation of only one alkyl group even though the active site contains a Cys residue from each of the two subunits. This result suggests that the enzyme shows half-site reactivity. The labeled enzyme was further subjected to exhaustive alkylation with unlabeled iodoacetate, permitting tryptic hydrolysis and isolation of an active site peptide in 30% yield. The specific radioactivity of the peptide was consistent with the first result, that only 1 mol of alkyl group was initially incorporated into active site. The active site peptide (14 residues) was sequenced and found to possess homology with the clostridial proline racemase.
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