Kinetic Analysis of the L-Ornithine Transcarbamoylase from Pseudomonas saVastanoi pv. phaseolicola That Is Resistant to the Transition State Analogue (R)-N δ -(N′-Sulfodiaminophosphinyl)-L-ornithine ² Matthew D. Templeton,* ,‡ Laurie A. Reinhardt, § Charles A. Collyer, | Robin E. Mitchell, ‡ and W. Wallace Cleland § Horticulture and Food Research Institute of New Zealand, Mt. Albert Research Centre, Auckland, New Zealand, Institute for Enzyme Research and Department of Biochemistry, UniVersity of Wisconsin, Madison, Wisconsin 53726, and School of Molecular and Microbial Biosciences, Sydney UniVersity, Sydney 2006, Australia ReceiVed December 7, 2004; ReVised Manuscript ReceiVed January 19, 2005 ABSTRACT:(R)-N δ -(N′-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas saVastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. saVastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding “SMG” loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the 13 C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k cat to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K i values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k cat . Pseudomonas saVastanoi pv. phaseolicola (synonym Pseudomonas syringae pv. phaseolicola) causes halo blight of French beans (Phaseolus Vulgaris L.). The disease symptoms include chlorosis around the site of infection that is caused by the diffusion of a toxin named phaseolotoxin. Phaseolotoxin has a highly unusual structure consisting of a tripeptide backbone of L-homoarginine, L-alanine, and L- ornithine (Orn), 1 and attached to the δ-amino group of Orn is an (R)-sulfodiaminophosphinyl residue (Figure 1a) (1, 2). In plants, the tripeptide is cleaved by peptidases to release (R)-N δ -(N′-sulfodiaminophosphinyl)-L-ornithine (PSorn); it is the most active component of the toxin (3) (Figure 1b). PSorn has been shown to be a potent active site directed inhibitor of the enzyme ornithine transcarbamoylase (OT- Case; E.C. 2.1.3.3) which binds to the enzyme from Escherichia coli ARGI, with a dissociation constant of 1.6 pM (4). OTCase catalyzes the condensation of carbamoyl phosphate (CP) and Orn to form L-citrulline and inorganic phosphate. In plants and microbes it is involved in arginine biosynthesis, whereas in mammals it is the first step of the urea cycle. OTCase has an ordered bi-bi mechanism where ² This research was supported by the Marsden Fund, Contract HRT801 to M.D.T. and R.E.M., and Grant GM 18938 from the National Institutes of Health to W.W.C. * To whom correspondence should be addressed at HortResearch, Private Bag 92-169, Auckland, New Zealand. Phone: +64-9-815-4200 ext 7316. Fax: +64-9-815-4201. E-mail: mtempleton@hortresearch.co.nz. ‡ Mt. Albert Research Centre. § University of Wisconsin. | Sydney University. 1 Abbreviations: ARGI, ornithine transcarbamoylase encoded by the Escherichia coli argI gene; AVA, 5-aminovaleric acid; Cf, forward commitment to catalysis; CP, carbamoyl phosphate; DAB, diaminobu- tane; DABA, diaminobutyric acid; DTT, dithiothreitol; EDTA, ethyl- enediaminetetraacetic acid; ESMS, electrospray mass spectrometry; IE, isotope effect; IPTG, isopropyl -D-thiogalactopyranoside; NAC, near attack conformers; Orn, L-ornithine; OTCase, ornithine transcarbam- oylase; PALO, N-(phosphonoacetyl)-L-ornithine; PCR, polymerase chain reaction; PMSF, phenylmethanesulfonyl fluoride; PSorn, (R)- N δ -(N′-sulfodiaminophosphinyl)-L-ornithine; ROTCase, OTCase en- coded by argK resistant to PSorn; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAPS, N-tris(hydroxym- ethyl)methyl-3-aminopropanesulfonic acid; Tris, tris(hydroxymethyl)- aminomethane; TS, transition state. 10.1021/bi047432x CCC: $30.25 © xxxx American Chemical Society PAGE EST: 7.5 Published on Web 00/00/0000