THE ATP-BINDING CASSETTE (ABC) TRANSPORTER FOR MALTOSE MALTODEXTRINS OF SALMONELLA-TYPHIMURIUM - CHARACTERIZATION OF THE ATPASE ACTIVITY ASSOCIATED WITH THE PURIFIED MALK SUBUNIT

The ATPase activity associated with the purified MalK subunit of the m altose transport complex of Salmonella typhimurium, a bacterial ATP-Bi nding Cassette (ABC) transporter (Walter, C., Honer zu Bentrup, K., an d Schneider, E. (1992) J. Biol. Chem. 267, 8863-8869), was characteriz ed in detail. The analysis of the kinetics of ATP hydrolysis yielded a K(m) value of 70 +/- 4 muM and a V(max) of 1.3 +/- 0.3 mumol/min/mg o f protein. Both GTP and CTP also served as substrates. While MalK exhi bited nearly the same affinity for GTP as for ATP, the Michaelis const ant for CTP as a substrate was much higher. ATP hydrolysis was strongl y dependent on the presence of Mg2+ ions. Mn2+ at low concentrations, but neither Ca2+ nor Zn2+ partially substituted for Mg2+. The ATPase a ctivity was optimal at slightly alkaline pH and was stimulated in the presence of both glycerol (7.5%) and dimethyl sulfoxide (Me2SO) (5%). ADP and the non-cleavable substrate analog ATPgammaS (adenosine 5'-O-( 3-thiotriphosphate)) were identified as competitive inhibitors. The Ma lK-ATPase was resistant to specific inhibitors of F-, P-, and V-type A TPases, such as dicyclohexylcarbodiimide, azide, vanadate, or bafilomy cin A1. In contrast, micromolar concentrations of the sulfhydryl reage nt N-ethylmaleimide strongly inhibited the enzymatic activity. This in hibition was blocked in the presence of ATP. These results suggest tha t the intrinsic ATPase activity of purified MalK can be clearly distin guished from other ATP-hydrolyzing enzymes, e.g. ion-translocating ATP ases.