Purification of ornithine carbamoyltransferase from kidney bean ( Phaseolus vulgaris L.) leaves and comparison of the properties of the enzyme from canavanine-containing and -deficient plants

Abstract

Kidney bean (Phaseolus vulgaris L.) ornithine carbamoyltransferase (OCT; EC 2.1.3.3) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonoacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 8540-fold-purified OCT exhibited a specific activity of 526 micromoles citrulline per minute per milligram of protein at 35 degrees C and pH 8.0. The enzyme represents approximately 0.01% of the total soluble protein in the leaf. The molecular mass of the native enzyme was approximately 109 kDa as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single band of molecular mass 36 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at a single isoelectric point of 6.6 when subjected to denaturing isoelectric focusing. These results suggest that the enzyme is a trimer of identical subunits. Among the tested amino acids, L-cysteine and S-carbamoyl-L-cysteine were the most effective inhibitors of the enzyme. The OCT of kidney bean showed a very low activity towards canaline. The OCTs of canavanine-deficient plants have very low canaline-dependent activities, but the OCTs of canavanine-containing plants showed high canaline-dependent activities. It was assumed that the substrate specificity of this enzyme determines the canavanine synthetic activity of the urea cycle.

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

“…Three (acetylornithine aminotransferase, ornithine carbamoyltransferase, and argininosuccinate lyase) are implicated in the synthesis of arginine, catalyzing the fourth, sixth, and eighth (last) step of the pathway, respectively (37). Interestingly, possibly reflecting activation by redox, sulfhydryl compounds were reported to increase the activity of ornithine carbamoyltransferase (38).…”

Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis

“…The degree of OTCase sequence conservation across all branches of the tree of life is quite high, with clearly recognizable sequence motifs for both carbamyl phosphate and ornithine binding. In addition to OTCase and ATCase, which are the best-characterized members of this superfamily, several transcarbamylases that catalyze the transfer of the carbamyl moiety to canaline, oxamate, or putrescine have been described (8,24,29). Extensive work on the evolutionary history of the transcarbamylase superfamily has been reported previously by Labedan et al (7).…”