To substantiate the functionality of a crystallographically evidenced allosteric site in Bacillus caldovelox arginase (Bewley et al., 1999), we have examined the kinetic consequences of the single mutations of Asp199 and Glu256, which interact with l-arginine in this site. The introduced mutations (Asp199 → Asn, Asp199 → Ala, Glu256 → Gln and Glu256 → Ala) had no effect on the hexameric structure of the enzyme (mol. wt. 195 ± 10 kDa). However, in contrast with the Michaelis-Menten kinetics exhibited by the wild-type species, the D199A, D199N, E256A and E256Q variants exhibited positive cooperativity with respect to l-arginine. The Glu278 → Ala mutation, which compromise interactions at the trimer-trimer interface, yielded trimeric species (mol. wt. 100 ± 5 kDa) exhibiting hyperbolic kinetics that changed to sigmoidal by the additional Glu256 → Ala mutation. In addition to demonstrating the total functionality of the trimer, our results also suggest that B. caldovelox is kinetically cooperative and that the commonly detected hyperbolic behavior results from binding of l-arginine as a typical allosteric activator.
Keywords: Allosteric site; Arginase; Bacillus caldovelox; Kinetics; Mutagenesis.
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