Kinetic and Structural Studies of Hydroxyproline 2-Epimerase

Abstract

Abstract Hydroxyproline 2-epimerase, purified to homogeneity from hydroxyproline-induced cells of Pseudomonas putida, behaved like a single peptide chain of approximately 64,000 molecular weight, based both on ultracentrifugal data and on a study of tryptic peptides and amino acid composition. Several methods indicated a total of 12 reduced cysteines per mole of native enzyme. Of these only six reacted with Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid)), including three which reacted relatively rapidly. Loss of enzyme activity was associated with reaction of one or more of the latter cysteines. The presence of substrate protected two of the rapidly reacting cysteines from reaction with Ellman's reagent and prevented loss of enzyme activity. A detailed kinetic study indicated that three 4-hydroxyproline epimers and two 3-hydroxyproline epimers had similar Km values; Vmax for the 3-hydroxyprolines was markedly lower than for the 4-hydroxyprolines. Vmax was reduced 2- to 3- fold by replacement of the α-hydrogen of substrates with deuterium or by substitution of deuterium oxide for water; deuterium substitution in both substrate and solvent had a cumulative effect in reducing Vmax. Nuclear magnetic resonance studies showed that the α-hydrogen exchanges during enzymatic racemization at the α-carbon, and that the rate of exchange parallels initial formation of product, consistent with a model in which two sites on the enzyme act as mutual, and perhaps concerted, acceptor and donor of the α-hydrogen in catalyzing epimerization. The indication of two cysteines in the active site suggests that these residues may act as the donor and recipient sites.