Uridine diphosphate d-glucuronic acid cyclase and uridine diphosphate d-glucuronic acid carboxy-lyase I from Lemna minor. Purification, characterization, and separation from uridine diphosphate d-glucuronic acid carboxy-lyase II

Abstract

Uridine 5′-(α- d-glucopyranosyluronic acid pyrophosphate) (UDPGluUA) cyclase [UDP GluUA → uridine 5′-(α- d-apio- d-furanosyl pyrophosphate) + CO 2] was purified 71-fold from Lemna minor by a four-step procedure. When this purification procedure was used some, but not all, of the UDPGluUA carboxy-lyase [UDPGluUA → uridine 5′-(α- d-xylopyranosyl pyrophosphate) + CO 2] was separated from UDPGluUA cyclase. The UDPGluUA carboxy-lyase that was separated from the UDPGluUA cyclase and the UDPGluUA carboxy-lyase that remained associated with it have completely different properties; these different forms of the enzyme have been given the names UDPGluUA carboxy-lyase II and I, respectively, and they are presumably isoenzymes. UDPGluUA cyclase and UDPGluUA carboxy-lyase I have K m values for UDPGluUA of 9.8 and 8.3 μ m, respectively. Both enzymes require the oxidized form of nicotinamide adenine dinucleotide for activity; for both, the concentration range for optimal activity is 1.0–2.0 m m. The reduced form of nicotinamide adenine dinucleotide and the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate cannot substitute for the oxidized form of nicotinamide adenine dinucleotide. Optimal activity for the two enzymes in 0.05 m potassium phosphate buffer is obtained at pH 7.8 and 7.7, respectively. Iodoacetamide and N-ethylmaleimide at 0.5 m m completely inhibit both enzymes. In the presence of 0, 0.01, and 0.05 m NH 4Cl, the relative activity of UDPGluUA cyclase was 100, 54, and 20%, respectively, while the relative activity of UDPGluUA carboxy-lyase I was 100, 283, and 379%, respectively. The same concentrations of N(CH 3) 4Cl either had no effect or inhibited both enzymes less than 20%. Neither enzyme is affected by 1 m m MgCl 2, MnCl 2, CaCl 2 ZnCl 2, CoCl 2, or NiCl 2 or by disodium ethylenediaminetetraacetate, 2,2′-bypridyl, or 8-hydroxyquinoline-5-sulfonic acid. In the presence of 0.1 and 0.2 m KCl, the relative activity of UDPGluUA cyclase was 75 and 62%, respectively, while the relative activity of UDPGluUA carboxy-lyase I was 67 and 49%, respectively. Almost identical results were obtained with NaCl. In these experiments the enzyme activities in the absence of salts were set equal to 100%.