Site-directed sulfhydryl labeling of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae: helix VIII comprises a portion of the sodium ion channel.
Helix VIII of the beta-subunit of the oxaloacetate decarboxylase of Klebsiella pneumoniae contains the functionally important residues betaN373, betaG377, betaS382, and betaR389. Using a functional oxaloacetate decarboxylase mutant devoid of Cys residues in the beta-subunit, each amino acid residue in helix VIII was replaced individually with Cys. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 20 single-Cys replacement mutants with methanethiosulfonate (MTS) reagents in the absence or presence of Na(+) ions. The pattern of accessibility of the MTS reagents from the periplasmic side of helix VIII shows a periodicity which suggests that this region is alpha-helical. In particular, a water-accessible face comprising betaN373, betaG377, betaS382, betaM386, and betaV390 may be part of a Na(+) channel. Cys residues introduced in the cytoplasmically oriented part of helix VIII were accessible to three different water-soluble MTS compounds and therefore believed to be exposed to water on this side of the membrane. Most residues located in the upper part of helix VIII (residues betaN373-betaV381C) were protected by Na(+) ions for inactivation by the MTS reagents. The distinct results on accessibility toward the different MTS reagents obtained in the presence or absence of Na(+) ions may suggest a conformational change upon binding of Na(+) in this region. The betaR389C mutant had a reduced activity and a pH optimum at pH 9, which could be restored to a wild-type pH optimum of 6.5 and to a 400% gain in activity upon chemical modification with 2-aminoethyl methanethiosulfonate.
Wild
Markus R
MR
Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
Pos
Klaas M
KM
Dimroth
Peter
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D002262:Carboxy-Lyases / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D007202:Indicators and Reagents; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008698:Mesylates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D007202:Indicators and Reagents; D008698:Mesylates; D021122:Protein Subunits; D015222:Sodium Channels; D013438:Sulfhydryl Compounds; C074502:methanethiosulfonate; D001120:Arginine; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine