Inactivation of Microbial Arginine Deiminases by l-Canavanine

Abstract

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.

Canavanine augments proapoptotic effects of arginine deprivation in cultured human cancer cells

“…In contrast, our preliminary data suggest that the effective canavanine dose increased considerably when the preparation of recombinant Mycoplasma hominis arginine deiminase was co-administered with canavanine, making this combination less feasible (data not shown). This may be explained by the fact that arginine deiminase can be efficiently inhibited by canavanine [46,49]. As the catalytic properties of pegylated recombinant human arginase do not differ significantly from those of the native human enzyme [9], our data suggest that canavanine is a suitable agent for combined treatment with arginase preparations and warrant further studies on its clinical potential.…”

“…However, in the case of a combination of arginine starvation with canavanine treatment, it should be taken into consideration that canavanine, as an arginine analogue, is also a substrate of these enzymes [44,45]. When a recombinant enzyme and the drug are administered simultaneously, this could lead to partial detoxification of canavanine, or, alternatively, canavanine, as the competitive arginine analogue, can inhibit activity of the enzymes that are mentioned [46,47]. However, we showed that at a 0.1 mmol/l concentration canavanine did not inhibit bovine liver arginase activity in vitro (Fig.…”

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

“…Crystallographically, MaAD was observed as a dimer [8] whereas the PaAD (shown in Fig. 3B) [29] and BcAD [30] were characterized as dimers of dimer. The GlAD C-terminal domain does not impede formation of the subunit-subunit interface of the dimer.…”

“…On the other hand, the reported crystal structures of MaAD, PaAD and BcAD [7,8,30] provided us with structural templates for the construction of a three-dimensional model of the GlAD N-terminal (“catalytic”) domain (residues 1–434). The GlAD model, shown in Fig.…”

“…5B) with the active sites of PaAD [7] (shown in Fig. 2A), BcAD [30] and MaAD [8] indicates that all substrate binding and catalytic residues as well as the catalytic scaffold on which they are positioned, are conserved. There is no indication that the entrance of the GlAD active site is less restricted than those of its bacterial counterparts.…”