Importance of conserved and variable C-terminal residues for the activity and thermal stability of the beta subunit of tryptophan synthase.
To assess the functional roles of helix 13 and of the conserved and variable residues in the C-terminal region (residues 378-397) of the tryptophan synthase beta subunit, we have constructed four C-terminal truncations and 12 point mutations. The effects of these mutations on kinetic and spectroscopic properties and thermal stability are reported here. The mutant beta subunits all form stable alpha2beta2 complexes that have been purified to homogeneity. The mutant alpha2beta2 complexes are divided into two classes on the basis of activity in the reaction of L-serine with indole to form tryptophan. Class I enzymes, which have mutations at Arg-379 or Asp-381 or truncations (384-397 or 385-397), exhibit significant activity (1-38% of wild type). Class II enzymes, which have mutations at Lys-382 or Asp-383 or truncations (382-397 or 383-397), exhibit very low activity (<1% of wild type). Although Class II enzymes have drastically reduced activity in the reaction of L-serine with indole and an altered distribution of enzyme-substrate intermediates in the reaction of L-serine with beta-mercaptoethanol, they retain activity in the reaction of beta-chloro-L-alanine with indole. Correlation of the results with the three-dimensional structure of the alpha2beta2 complex suggests that Lys-382 and Asp-383 serve important roles in a proposed "open" to "closed" conformational change that occurs in the reactions of L-serine. Because mutant beta subunits having C-terminal truncations (383-397 or 384-397) undergo much more rapid thermal inactivation at 60 degrees C than the wild type beta subunit, the C-terminal helix 13 stabilizes the beta subunit.
Yang
L
L
Enzyme Structure and Function Section, Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Ahmed
S A
SA
Rhee
S
S
Miles
E W
EW
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012694:Serine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
D015091:beta-Alanine; C006977:3-chloroalanine; D012694:Serine; D014367:Tryptophan Synthase