Functional Characterization of a Novel ArgA from Mycobacterium tuberculosis

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Abstract
The Mycobacterium tuberculosis gene Rv2747 encodes a novel 19-kDa ArgA that catalyzes the initial step in l-arginine biosynthesis, namely the conversion of l-glutamate to α-N-acetyl-l-glutamate. Initial velocity studies reveal that Rv2747 proceeds through a sequential kinetic mechanism, with Km values of 280 mM for l-glutamine and 150 μM for acetyl-coenzyme A and with a kcat value of 200 min−1. Initial velocity studies with l-glutamate showed that even at concentrations of 600 mM, saturation was not observed. Therefore, only a kcat/Km value of 125 M−1 min−1 can be calculated. Inhibition studies reveal that the enzyme is strongly regulated by l-arginine, the end product of the pathway (50% inhibitory concentration, 26 μM). The enzyme was completely inhibited by 500 μM arginine, with a Hill coefficient of 0.60, indicating negatively cooperative binding of l-arginine.