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Biochem J. Dec 1, 1984; 224(2): 379–388.
PMCID: PMC1144443

The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin.

Abstract

The mechanism of inhibition of ornithine transcarbamoylase by the bacterial toxin phaseolotoxin [N-delta-(phosphosulphamyl)ornithylalanylhomoarginine] was investigated. Ornithine transcarbamoylase was purified by affinity chromatography from Escherichia coli W argR- by using N-delta-(phosphonoacetyl)ornithine as the ligand. Under steady-state conditions phaseolotoxin inhibition was reversible and exhibited mixed kinetics with respect to carbamoyl phosphate. The apparent Ki and apparent K'i were 0.2 microM and 10 microM respectively. Inhibition with respect to ornithine was noncompetitive, with an apparent Ki of 0.9 microM. These data are consistent with competitive binding of phaseolotoxin to the carbamoyl phosphate-binding site of the enzyme. The toxin also appears to be able to bind to the enzyme-carbamoyl phosphate complex, although, since K'i is 50 times greater than Ki, this event is kinetically much less significant. In the presence of phaseolotoxin ornithine transcarbamoylase exhibited a transient phase of activity before a steady state. This is consistent with low rates of association and dissociation for the toxin with enzyme and the enzyme-toxin complex. Rate constants of 2.5 X 10(4)M-1 X s-1 and 5 X 10(-3)s-1 were estimated for the association and dissociation constants respectively.

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Selected References

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  • Benson JR. Improved ion-exchange resins. Methods Enzymol. 1977;47:19–31. [PubMed]
  • Boyde TR, Rahmatullah M. Optimization of conditions for the colorimetric determination of citrulline, using diacetyl monoxime. Anal Biochem. 1980 Sep 15;107(2):424–431. [PubMed]
  • Cha S. Tight-binding inhibitors-I. Kinetic behavior. Biochem Pharmacol. 1975 Dec 1;24(23):2177–2185. [PubMed]
  • Cornish-Bowden A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem J. 1974 Jan;137(1):143–144. [PMC free article] [PubMed]
  • DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. [PMC free article] [PubMed]
  • Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. [PubMed]
  • Hoogenraad NJ. Synthesis and properties of delta-N-(phosphonacetyl)-L-ornithine. A transition-state analog inhibitor of ornithine transcarbamylase. Arch Biochem Biophys. 1978 May;188(1):137–144. [PubMed]
  • Hoogenraad NJ, Sutherland TM, Howlett GJ. Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog. Anal Biochem. 1980 Jan 1;101(1):97–102. [PubMed]
  • Kalousek F, François B, Rosenberg LE. Isolation and characterization of ornithine transcarbamylase from normal human liver. J Biol Chem. 1978 Jun 10;253(11):3939–3944. [PubMed]
  • Kwok OC, Ako H, Patil SS. Inactivation of bean ornithine carbamoyltransferase by phaseotoxin: effect of phosphate. Biochem Biophys Res Commun. 1979 Aug 28;89(4):1361–1368. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Legrain C, Stalon V. Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis. Eur J Biochem. 1976 Mar 16;63(1):289–301. [PubMed]
  • Legrain C, Stalon V, Noullez JP, Mercenier A, Simon JP, Broman K, Wiame JM. Structure and function of ornithine carbamoyltransferases. Eur J Biochem. 1977 Nov 1;80(2):401–409. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Lusty CJ, Jilka RL, Nietsch EH. Ornithine transcarbamylase of rat liver. Kinetic, physical, and chemical properties. J Biol Chem. 1979 Oct 25;254(20):10030–10036. [PubMed]
  • MAAS WK. Studies on repression of arginine biosynthesis in Escherichia coli. Cold Spring Harb Symp Quant Biol. 1961;26:183–191. [PubMed]
  • Marshall M, Cohen PP. Ornithine transcarbamylase from Streptococcus faecalis and bovine liver. I. Isolation and subunit structure. J Biol Chem. 1972 Mar 25;247(6):1641–1653. [PubMed]
  • Mitchell RE, Bieleski RL. Involvement of phaseolotoxin in halo blight of beans: transport and conversion to functional toxin. Plant Physiol. 1977 Nov;60(5):723–729. [PMC free article] [PubMed]
  • Nimmo HG, Nimmo GA. A general method for the localization of enzymes that produce phosphate, pyrophosphate, or CO2 after polyacrylamide gel electrophoresis. Anal Biochem. 1982 Mar 15;121(1):17–22. [PubMed]
  • NOVICK RP, MAAS WK. Control by endogenously synthesized arginine of the formation of ornithine transcarbamylase in Escherichia coli. J Bacteriol. 1961 Feb;81:236–240. [PMC free article] [PubMed]
  • Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. [PubMed]
  • Staskawicz BJ, Panopoulos NJ, Hoogenraad NJ. Phaseolotoxin-insensitive ornithine carbamoyltransferase of Pseudomonas syringae pv. phaseolicola: basis for immunity to phaseolotoxin. J Bacteriol. 1980 May;142(2):720–723. [PMC free article] [PubMed]
  • Sullivan PA, Soon CY, Schreurs WJ, Cutfield JF, Shepherd MG. The structure of L-lactate oxidase from Mycobacterium smegmatis. Biochem J. 1977 Aug 1;165(2):375–383. [PMC free article] [PubMed]
  • Tam LQ, Patil SS. Mode of Action of the Toxin from Pseudomonas phaseolicola: II. Mechanism of Inhibition of Bean Ornithine Carbamoyltransferase. Plant Physiol. 1972 May;49(5):808–812. [PMC free article] [PubMed]
  • Williams JW, Morrison JF. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 1979;63:437–467. [PubMed]

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