Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis

Aminoimidazole ribonucleotide (AIR) synthetase (PurM) catalyzes the convers ion of formyl-glycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and Pi , the fifth step in de novo purine biosynthesis. The ATP binding domain of the E. coli enzyme has been investigated using the affinity label [C-14]-p- fluorosulfonylbenzoyl adenosine (FSBA). This compound results in time-depen dent inactivation of the enzyme which is accelerated by the presence of FGA M, and gives a K-i = 25 mu M and a k(inact) = 5.6 x 10(-2) min(-1). The ina ctivation is inhibited by ADP and is stoichiometric with respect to AIR syn thetase. After trypsin digestion of the labeled enzyme, a single labeled pe ptide has been isolated, I-X-G-V-V-K, where X is Lys27 modified by FSBA. Si te-directed mutants of AIR synthetase were prepared in which this Lys27 was replaced with a Gin, a Leu, and an Arg and the kinetic parameters of the m utant proteins were measured. All three mutants gave k(cats) similar to the wild-type enzyme and K(m)s for ATP less than that determined for the wild- type enzyme. Efforts to inactivate the chicken liver trifunctional AIR synt hetase with FSBA were unsuccessful, despite the presence of a Lys27 equival ent. The role of Lys27 in ATP binding appears to be associated with the met hylene linker rather than its E-amino group. The specific labeling of the a ctive site by FSBA has helped to define the active site in the recently det ermined structure of AIR synthetase [Li, C., Kappock, T. J., Stubbe, J., We aver, T. M., and Ealick, S. E. (1999) Structure (in press)], and suggests a dditional flexibility in the ATP binding-region.