Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Campylobacter jejuni

Figures & data

Table I.  Kinetic parameters for dUTP and dUDP hydrolysis by Campylobacter jejuni dUTPase.

Substrate	Km(μM)	Ki(μM)	kcat(s^− 1)	kcat/Km(M^− 1 s^− 1)
dUTP	0.66	11.4	12.3	18.6 × 10^6
dUDP	4.74	50.0	16.6	3.5 × 10^6

Table II.  Kinetic parameters for dUTP hydrolysis accomplished by monomeric, dimeric and trimeric dUTPases.

Organism (Reference)	Structure	Km(μM)	kcat (s^− 1)	kcat/Km (M^− 1s^− 1)	dUMP: Kip (μM)
C. jejuni	dimer	0.66	12.3	18.6 × 10^6	11.4
E. coli [20]	trimer	0.2	7.4	3.0 × 10^7	1500
EIAV[21]	trimer	1.1	25	2.0 × 10^7	130
MMTV[22]	trimer	0.8	1.5	1.9 × 10^6	nd
HSV-1[23]	monomer	0.3	12	4.0 × 10^7	170
L. major[15]	dimer	2.1	49	2.3 × 10^7	13.1
T.cruzi[9]	dimer	0.5	2.8	5.2 × 10^6	18.4

Figure 1.  Complete hydrolysis of dUTP by dUTPase under multiple-turnover conditions. The inset shows the linear transformation of the data between the arrows, according to the integrated Michaelis-Menten equation adapted for enzymes with product inhibition, and the corresponding regression line. The reaction was recorded at 573 nm after reacting 30 nm dUTPase and 50 μM dUTP in the presence of 25 mM MgCl₂, 2M BICINE, and 50 μM cresol red at pH 8.

Figure 2.  Inhibition by dUMP of dUTP hydrolysis. The K_i value for product (dUMP) inhibition was calculated from the K_mapp values obtained at different dUMP concentrations (5 to 100 μM). The K_i for dUMP was 11.4 μM and the real K_m 0.66 μM.

Figure 3.  Inhibition by dUMP of dUDP hydrolysis. The K_i value for product (dUMP) inhibition was calculated with the K_mapp value obtained at different dUMP concentrations (5 to 100 μM). The K_i for dUMP was 50 μM and the real K_m 4.74 μM.

Table III.  Inhibition constants K_i (μM) for compounds against Campylobacter jejuni, P. falciparum and human dUTPases.

STRUCTURE	Ki(μM) Human^*	Ki(μM) Campylobacter jejuni	Ki(μM) Plasmodium falciparum^*
	135	>1 mM	238
	156	>1 mM	1.9
	298	547	57.7
	350	>1 mM	255.7
	>1 mM	>1 mM	1.16
	>1 mM	>1 mM	628
	807	>1 mM	89.39
	908	>1 mM	2.8
	805	>1 mM	4.2
	>1 mM	>1 mM	227.1
	>1 mM	>1 mM	110.7

* Values previously reported[18].

G Larsson, PO Nyman, and JO Kvassman. (1996). Kinetic characterization of dUTPase from Escherichia coli. J Biol Chem 271 (39):24010–24016.

 PubMedGoogle Scholar

J Nord, G Larsson, JO Kvassman, AM Rosengren, and PO Nyman. (1997). dUTPase from the retrovirus equine infectious anemia virus: specificity, turnover and inhibition. FEBS Lett 414 (2):271–274.

 PubMedGoogle Scholar

O Bjornberg, and PO Nyman. (1996). The dUTPases from herpes simplex virus type 1 and mouse mammary tumour virus are less specific than the Escherichia coli enzyme. J Gen Virol 77 ((Pt 12)):3107–3111.

 PubMedGoogle Scholar

AC Bergman, PO Nyman, and G Larsson. (1998). Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1. FEBS Lett 441 (2):327–330.

 PubMedGoogle Scholar

F Hidalgo-Zarco, AG Camacho, V Bernier-Villamor, J Nord, LM Ruiz-Perez, and D Gonzalez-Pacanowska. (2001). Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Leishmania major. Protein Sci 10 (7):1426–1433.

 PubMedGoogle Scholar

V Bernier-Villamor, A Camacho, F Hidalgo-Zarco, J Perez, LM Ruiz-Perez, and D Gonzalez-Pacanowska. (2002). Characterization of deoxyuridine 5′-triphosphate nucleotidohydrolase from Trypanosoma cruzi. FEBS Lett 526 (1–3):147–150.

 PubMedGoogle Scholar

C Nguyen, G Kasinathan, I Leal-Cortijo, A Musso-Buendia, M Kaiser, R Brun, LM Ruiz-Perez, NG Johansson, D Gonzalez-Pacanowska, and IH Gilbert. (2005). Deoxyuridine triphosphate nucleotidohydrolase as a potential antiparasitic drug target. J Med Chem 48 (19):5942–5954.

 PubMedGoogle Scholar