The ATP-binding cassette (ABC) transporter for maltose/maltodextrins of Salmonella typhimurium. Characterization of the ATPase activity associated with the purified MalK subunit.

S Morbach,S Tebbe,E Schneider

doi:10.1016/s0021-9258(17)46673-7

Abstract

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.

Highlights

The ATPaseactivity associated with the purMifiaeldK of the substrateacross the membraneby a still unknown mechsubunit of the maltose transport complexSaolfmonella anism
The maltose transport system of S . typhirnurium and other enterobacteria is a member of the ABC transporter family [11]
The protein complex that translocates the substrateacross the cytoplasmic membrane is composed of onecopy each of the integral membrane proteins MalF (57kDa) and MalG (32m a ), and two copiesof the peripherally bound ATP-bindingsubunit MalK (40 kDa) [7].Incorporated into liposomes, the MalFGK, complex catalyzed ATP hydrolysis only in the presence of the substrate-loaded binding protein MalE, whichin thebacterial cell is located in theperiplasm [7].This observation was interpreted to mean that theATPase activity of MalK is blocked by interaction with MalF and/or MalG, unless a conformational change initiated by the liganded binding protein “unlocks”the enzymatic activity

Summary

CHARACTERIZATION OF THE ATPase ACTMTY ASSOCIATED WITH THE PURIFIED MalK SUBUNIT*

From the ArbeitsaruDDe Mikrobiolopie, Fachbereich BiologielChemie, Universitat Osnabruck, I ". As indicated by a n 8-fold higher Michaelis constant (542 w) Glycerol and MezSO Stimulate ATPase Activity-Glycerol (Fig. lb) This result is consistent with the recent finding thathas been found to stabilize nucleotide binding folds in proteins only GTP effectively competed with ATP for the nucleotide (see, e.g., Ref. 29) and was added to all buffers binding siteon MalK [17]. The ATPase assay used in the present study requires a severalfold dilution of MalK in assay buffer, resulting in a residual glycerol concentration of0.8% Under these conditions the activity of MalK was low ADP and ATPyS Are Competitive Inhibitors of MalK-The. ATPase activity of MalK was recorded in the presence of increasing concentrations ofADP and of the non-hydrolyzable substrate analog ATPyS, respectively. The data area combinationof different experiments in which the 100%value varied between 0.5and 1.5 pmol of PJmirdmg

ATPase activity

Findings

DISCUSSION