Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase:: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His(68) and His(89) from one and His(141) from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry, 39, 13336-13343]. Glu(275) is 2.8 Angstrom from His(141) in the ASL crystal structure, and Lys(268) is also in the active site region; Glu(275) and Lys(268) come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys(268) by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu(275) was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys(268) mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu(275) mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-H-3]-adenylosuccinate reveal that none of the Glu(275) mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys(268) participates in binding substrate and that Glu(275) is essential for catalysis because of its interaction with His(141). Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.