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 maltose transport complex of Salmonella typhimurium, a bacterial ATP-Binding Cassette (ABC) transporter (Walter, C., Höner zu Bentrup, K., and Schneider, E. (1992) J. Biol. Chem. 267, 8863-8869), was characterized in detail. The analysis of the kinetics of ATP hydrolysis yielded a Km value of 70 +/- 4 microM and a Vmax of 1.3 +/- 0.3 mumol/min/mg of protein. Both GTP and CTP also served as substrates. While MalK exhibited nearly the same affinity for GTP as for ATP, the Michaelis constant for CTP as a substrate was much higher. ATP hydrolysis was strongly dependent on the presence of Mg2+ ions. Mn2+ at low concentrations, but neither Ca2+ nor Zn2+ partially substituted for Mg2+. The ATPase activity 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 ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) were identified as competitive inhibitors. The MalK-ATPase was resistant to specific inhibitors of F-, P-, and V-type ATPases, such as dicyclohexylcarbodiimide, azide, vanadate, or bafilomycin A1. In contrast, micromolar concentrations of the sulfhydryl reagent N-ethylmaleimide strongly inhibited the enzymatic activity. This inhibition was blocked in the presence of ATP. These results suggest that the intrinsic ATPase activity of purified MalK can be clearly distinguished from other ATP-hydrolyzing enzymes, e.g. ion-translocating ATPases.
Morbach
S
S
Arbeitsgruppe Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Federal Republic of Germany.
Tebbe
S
S
Schneider
E
E
D018528:ATP-Binding Cassette Transporters; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000378:metabolism; D004121:Dimethyl Sulfoxide; D005033:Ethylmaleimide / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D012486:Salmonella typhimurium / Q000737:chemistry*; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; C068021:MalK protein, Bacteria; C489399:MalK protein, Salmonella typhimurium; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium; D005033:Ethylmaleimide; D005990:Glycerol; D004121:Dimethyl Sulfoxide