Separation of Ornithine and Lysine Activities of the Ornithine-Transcarbamylase-Catalyzed Reaction

Abstract

Several lines of evidence are presented that an entity is present in mitochondria which converts lysine and carbamylphosphate to homocitrulline, distinct from the ornithine transcarbamylase (OTC): lack of inhibition by lysine of OTC with ornithine as substrate at the pH of the mitochondrial matrix, a K(m) for lysine of 6.3 mmol/l in the crude mitochondrial extract, versus a K(m) of 55.3 mmol/l after partial purification of OTC, decrease of the specific activity of OTC with lysine as substrate and increase of the specific activity with ornithine as substrate after heat denaturation, and separation of the activities for ornithine and lysine after isoelectric focusing. This would explain the excretion of homocitrulline in the HHH-syndrome, saccharopinuria, citrullinemia and hyperlysinemia. The fact that homocitrulline excretion is not observed in OTC deficiency suggests that the activity responsible for homocitrulline excretion is closely related to OTC.

Further Evidence for a Separate Enzymic Entity for the Synthesis of Homocitrulline, Distinct from the Regular Ornithine Transcarbamylase

“…The ratio of transcarbamylase activities for orni thine and lysine is thus not constant, which would be expected if the same enzyme spe cies was responsible for the reaction with either substrate. A separate enzymic entity must therefore be present, distinct from the 'regular' OTC, in agreement with earlier ob servations [14], The observation that this en zymic entity is released ahead of OTC sug gests a different localization. Similar results were obtained with mitochondria from rat liver and from beef liver.…”

“…Neither mouse nor rat liver mitochondrial OTC showed the heterogeneity as was ob served with the purified OTC from bovine liver [23,24] or with extracts of bovine liver mitochondria [14]. Such heterogeneity is, however, observed in extracts of bovine liver mitochondria by the high-performance liq uid chromatography technique, as is illus trated in figure 6.…”

“…An extract prepared at a digitonin to protein ratio of 0.4 showed the major portion of the OTC to be eluted at 80% of the graheterogeneity [18,22]. Heterogeneity for the lysine activity of bovine OTC has likewise been demonstrated by isoelectric focusing, with the lysine activity separated from the ornithine activity [14], To investigate this further high-performance liquid chromatog raphy was used in an attempt to separate multiple forms of OTC. The results of such an experiment for the unaffected male sparse fur mouse is shown in figure 4a.…”

“…Evidence has been presented that the HHH syndrome is dence, [cf. 13,16], or the localization of the activity converting lysine and carbamylphosphate to homocitrulline [ 14] is different from that for the conversion of ornithine and carbamylphosphate to citrulline. The present communication describes experiments ad dressing this problem.…”

Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes

“…This group of proteins includes aspartate transcarbamylase (ATCase ; EC 2.1.3.2), OTCase (EC 2.1.3.3), oxamate transcarbamylase (EC 2.1.3.5) [1,2], putrescine transcarbamylase (EC 2.1.3.6) [3,4] and the putative lysine transcarbamylase (EC 2.1.3.8) [5,6]. These enzymes share a common biological function ; to transfer a carbamoyl group from carbamoyl phosphate (CP) to an amino group of the second substrate.…”