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Effect of mutations at Met‐88 and Met‐90 on the biotination of Lys‐89 of the apo 1.3S subunit of transcarboxylase
Author(s) -
Shenoy Bhami C.,
Paranjape Suvinay,
Murtif Vicki L.,
Kumar Ganesh K.,
Samols David,
Wood Harland G.
Publication year - 1988
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2.9.3131174
Subject(s) - mutant , protein subunit , site directed mutagenesis , mutagenesis , microbiology and biotechnology , biology , wild type , escherichia coli , biochemistry , propionibacterium , gene , genetics , bacteria
The apo 1.3S subunit of transcarboxylase contains the sequence Ala‐87‐Met‐88‐Lys‐89‐Met‐90, and it is Lys‐89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl‐CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met‐88 and Met‐90 in specifying Lys‐89 for biotination by synthetase was examined by site‐directed mutagenesis. Genes of the 1.3S subunit coding for Thr‐88, Leu‐88, or Leu‐90 were generated by oligonucleotide‐directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild‐type subunit. The V max for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met‐88 or Met‐90, the K m for the mutant was lower than that of the wild‐type or mutant Thr‐88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr‐88 and Leu‐90 was biotinated than with the wild‐type or mutant Leu‐88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met‐88 and Met‐90 are not required for biotination of the apo 1.3S subunit by the synthetases.—S henoy , B. C.; P aranjape , S.; M urtif , V. L.; K umar , G. K.; S amols , D; W ood , H. G. Effect of mutations at Met‐88 and Met‐90 on the biotination of Lys‐89 of the apo 1.3S subunit of transcarboxylase. FASEB J. 2: 2505‐2511; 1988.