Partial revertants of tryptophan synthetase alpha chain active site mutant Asp60-->Asn.
Residue Asp60 of the tryptophan synthetase alpha chain of Escherichia coli is though to interact with the pyrrole NH of substrate indole-3-glycerol phosphate and facilitate its cleavage to indole and glyceraldehyde 3-phosphate. Two distinguishable partial revertants of DN60 tryptophan synthetase alpha mutant trpA34 were analyzed. The slower growing partial revertant, PR1, had the second-site change, YD102. The other partial revertant, PR2, lacked three consecutive base pairs, resulting in replacement of Ala59 and Asn60 of the DN60 mutant alpha polypeptide by Asp. Inspection of the three-dimensional structure of the enzyme-substrate analog complex revealed that Tyr102 is in the vicinity of the pyrrole NH of the substrate. The PR1 alpha chain has a near normal Km for substrate, whereas the PR2 polypeptide has greatly reduced substrate affinity. The PR2 polypeptide is more active than the PR1 polypeptide in the alpha beta reaction in vitro and appears to be more active than the PR1 polypeptide in vivo. Attempts to obtain repeat occurrences of the PR2 deletion mutation were unsuccessful. A third type of trpA34 partial revertant, PR3, that grows very poorly in minimal medium, also has a Tyr102 replacement: YF102. These findings demonstrate that each of the second-site mutations affects a residue located in the vicinity of the active site residue altered by the primary mutation. Slightly leaky mutant trpA89, genetically altered near the site of the trpA34 mutation, was found to have a GS61 substitution.
Yanofsky
C
C
Department of Biological Sciences, Stanford University, California 94305-5020.
Yee
M C
MC
Horn
V
V
D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D001216:Asparagine; D014367:Tryptophan Synthase