An OxlT homology model suggests R272 and K355 in transmembrane heli
ces 8 and 11,respe
ctively, are
criti
cal to OxlT-mediated transport. We offer positive eviden
ce supporting this idea bystudying OxlT fun
ction after
cysteine residues were separately introdu
ced at these positions. Withoutfurther treatment, both mutant proteins had a null phenotype when they were re
constituted intoproteoliposomes. By
contrast, signifi
cant re
covery of fun
ction o
ccurred when proteoliposomes were treatedwith MTSEA (methanethiosulfonate ethylamine), a thiol-spe
cifi
c reagent that implants a positively
chargedamino group. In ea
ch
case, there was a 2-fold in
crease in the Mi
chaelis
constant (
KM) for oxalate self-ex
change (from 80 to 160
![](/images/entities/mgr.gif)
M), along with a 5-fold (K355C) or 100-fold (R272C) redu
ction in
Vmaxcompared to that of the
cysteine-less parental protein. Analysis by MALDI-TOF
confirmed that MTSEAintrodu
ced the desired modifi
cation. We also examined substrate sele
ctivity for the treated derivatives.While oxalate remained the preferred substrate, there was a shift in preferen
ce among other substrates sothat the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate> formate > malonate). This shift is
consistent with the idea that the substrate-binding site is redu
ced insize via introdu
ction of the SCH
2CH
2NH
3+ addu
ct, whi
ch generates a side
chain that is ~1.85 Å longerthan that of lysine or arginine. These findings lead us to
con
clude that R272 and K355 are essential
components of the OxlT substrate-binding site.