Functional Characterization of a Novel ArgA from Mycobacterium tuberculosis

Abstract

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 K m values of 280 mM for l -glutamine and 150 μM for acetyl-coenzyme A and with a k cat 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 k cat / K m 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.