Expression, purification and kinetic characterization of wild-type human ornithine transcarbamylase and a recurrent mutant that produces 'late onset' hyperammonaemia

Hiroki Morizono, Mendel Tuchman, Basavapatna S. Rajagopal, Mark T. McCann, Chad D. Listrom, Xiaoling Yuan, Divakaramenon Venugopal, George Barany, Norma M. Allewell

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

Ornithine Transcarbamylase Deficiency, an X-linked disorder, is the most common cause of inherited urea cycle disorders. Approx. 90 mutations that produce reduced levels of ornithine transcarbamylase (OTCase) activity have been identified in patients. A model of the three-dimensional structure of OTCase, developed on the basis of its homology to the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase), and in good agreement with the crystal structure of Pseudomonas aeruginosa OTCase, indicates that many mutations that produce severe clinical symptoms are at the active site or buried in the interior of the protein. However, one of the few recurrent mutations, R277W, an alteration that produces a milder phenotype of ornithine transcarbamylase deficiency, is located in the model in a loop remote from the active site that is analogous to a similar loop (the 240's loop, a flexible loop of the catalytic chain of Escherichia coli aspartate transcarbamylase, comprised of residues 230-250) of ATCase. Human wild-type OTCase and the R277W mutant have been cloned and over-expressed in E. coli and a rapid and efficient purification method utilizing the bisubstrate analogue, N(δ)-(phosphonacetyl)-L-ornithine, has been developed and used to purify both proteins. Gel chromatography indicates both are trimeric. The pH dependence of the kinetic parameters of the wild-type enzyme is similar to that of E. coli OTCase, suggesting that its catalytic mechanism is similar, although its maximal activity is approx. 10-fold less. Compared with the wild-type, the R277W mutant has nearly 70-fold lower affinity for L-ornithine, shows no substrate inhibition, and its thermal stability is reduced by 5°C. Its reduced affinity for L-ornithine, which in turn results in lower activity at physiological concentrations of ornithine, as well as its reduced stability, may contribute to the clinical effects that it produces.

Original languageEnglish (US)
Pages (from-to)625-631
Number of pages7
JournalBiochemical Journal
Volume322
Issue number2
DOIs
StatePublished - Mar 1 1997

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