STRUCTURE AND FUNCTION OF SALMONELLA-TYPHIMURIUM OROTATE PHOSPHORIBOSYLTRANSFERASE - PROTEIN COMPLEMENTATION REVEALS SHARED ACTIVE-SITES

A solvent-exposed loop, comprising residues 98-119 of S. typhimurium o rotate phosphoribosyltransferase (OPRTase), is at the subunit interfac e of the dimeric enzyme, and its amino acid side chains potentially co ntact active sites on either subunit. A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme . OMP [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemi stry 33, 1287-1294] and enzyme . PRPP . orotate complexes [Scapin, G., Ozturk, D. H., Grubmeyer, C., and Sacchettini, J. C. (1995) Biochemis try 34, 10744-10754]. Lys-103, which is essential for activity [Ozturk , D. H., Dorfman, R. H., Scapin, G., Sacchettini, J. C., and Grubmeyer , C. (1995) Biochemistry 34, 10755-10763], may thus be functional in t he active site formed by the adjacent subunit. Asp-125 is an essential residue that is in the middle of the active site. Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produce d approximately 21-23% of the enzymatic activity of the wild-type OPRT ase. Heterodimer formation in the complemented mixtures was evidenced by various physical methods. Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit. As pr edicted from the three-dimensional structure, increased activity resul ting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtu res of D125N and either K73A or K73Q mutants. Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme. A K103A/D125N double mutant enzyme was also constructed and was able t o inactivate wild-type enzyme. Negative complementation between the wi ld-type and double mutant enzyme was used to determine the subunit dis sociation rate for the wild-type dimer, k(D) = 0.013 min(-1).