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Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase. 1989

M Merola, and A Martínez del Pozo, and H Ueno, and P Recsei, and A Di Donato, and J M Manning, and K Tanizawa, and Y Masu, and S Asano, and H Tanaka
Rockefeller University, New York, New York 10021.

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D003545 Cysteine A thiol-containing non-essential amino acid that is oxidized to form CYSTINE. Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004228 Dithionitrobenzoic Acid A standard reagent for the determination of reactive sulfhydryl groups by absorbance measurements. It is used primarily for the determination of sulfhydryl and disulfide groups in proteins. The color produced is due to the formation of a thio anion, 3-carboxyl-4-nitrothiophenolate. 5,5'-Dithiobis(2-nitrobenzoic Acid),DTNB,Ellman's Reagent,5,5'-Dithiobis(nitrobenzoate),Acid, Dithionitrobenzoic,Ellman Reagent,Ellmans Reagent,Reagent, Ellman's
D005998 Glycine A non-essential amino acid. It is found primarily in gelatin and silk fibroin and used therapeutically as a nutrient. It is also a fast inhibitory neurotransmitter. Aminoacetic Acid,Glycine, Monopotassium Salt,Glycine Carbonate (1:1), Monosodium Salt,Glycine Carbonate (2:1), Monolithium Salt,Glycine Carbonate (2:1), Monopotassium Salt,Glycine Carbonate (2:1), Monosodium Salt,Glycine Hydrochloride,Glycine Hydrochloride (2:1),Glycine Phosphate,Glycine Phosphate (1:1),Glycine Sulfate (3:1),Glycine, Calcium Salt,Glycine, Calcium Salt (2:1),Glycine, Cobalt Salt,Glycine, Copper Salt,Glycine, Monoammonium Salt,Glycine, Monosodium Salt,Glycine, Sodium Hydrogen Carbonate,Acid, Aminoacetic,Calcium Salt Glycine,Cobalt Salt Glycine,Copper Salt Glycine,Hydrochloride, Glycine,Monoammonium Salt Glycine,Monopotassium Salt Glycine,Monosodium Salt Glycine,Phosphate, Glycine,Salt Glycine, Monoammonium,Salt Glycine, Monopotassium,Salt Glycine, Monosodium
D000637 Transaminases A subclass of enzymes of the transferase class that catalyze the transfer of an amino group from a donor (generally an amino acid) to an acceptor (generally a 2-keto acid). Most of these enzymes are pyridoxyl phosphate proteins. (Dorland, 28th ed) EC 2.6.1. Aminotransferase,Aminotransferases,Transaminase
D001407 Bacillus A genus of BACILLACEAE that are spore-forming, rod-shaped cells. Most species are saprophytic soil forms with only a few species being pathogenic. Bacillus bacterium
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining

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