Properties of Allantoinase From Whole Developing Fruits of French Bean, Phaseolus vulgaris L

Abstract

In ureide-transporting legumes such as French bean, Phaseolus vulgaris L., allantoin is a major transport form of biologically fixed nitrogen. Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyses the conversion of allantoin to allantoate. In developing French bean fruits, seed coats contained the highest levels of soluble activity on a fresh weight basis, suggesting that this organ may be important in ureide metabolism. However, over 70% of the total allantoinase activity of the developing fruit was in the pods.

Allantoinase from whole developing French bean fruits was partially purified and characterised. Dithiothreitol inhibited activity, suggesting that disulfide bridges are necessary for activity. Mn²⁺ was found to relieve this inhibition to some extent; however, the presence of Mn²⁺ or EDTA in incubation mixtures did not affect activity. This suggests that, whilst allantoinase may not be a metalloenzyme, Mn²⁺ may have a role in maintaining the structure of the active enzyme. The K_m for allantoin was determined to be 40.3 mM and the molecular mass of allantoinase was 50 000. Acid pretreatment of a crude extract reduced activity to 82% of control activity.

On the basis of sucrose density fractionation of French bean fruit extracts, allantoinase was determined to be associated with the microsomal fraction of the cell, probably the endoplasmic reticulum, as activity was coincident with NADPH-cytochrome c reductase.