Lysine 87 in the beta subunit of tryptophan synthase that forms an internal aldimine with pyridoxal phosphate serves critical roles in transimination, catalysis, and product release.
This study provides valuable insights into the functions of the lysine residue that forms an internal aldimine with pyridoxal phosphate in the beta subunit of tryptophan synthase from Salmonella typhimurium. Our spectroscopic and kinetic studies demonstrate that a mutant alpha 2 beta 2 complex having beta subunit lysine 87 replaced by threonine forms external aldimines with several amino acids including L-serine, beta-chloro-1-alanine, L-tryptophan, and D-tryptophan. Because the rates of aldimine formation are very slow, we conclude that one role of lysine 87 in the wild type enzyme is to facilitate formation of external aldimines by transimination. Lysine 87 is an essential catalytic residue because the mutant alpha 2 beta 2 complex has no measurable activity in reactions catalyzed by the beta subunit and does not convert external aldimines to products. The mutant enzyme carries out two slow partial beta-elimination reactions: the conversion of beta-chloro-L-alanine and L-serine to enzyme-bound aminoacrylate. The reaction with L-serine is catalyzed by ammonia, which partially replaces the deleted epsilon-amino group. Lysine 87 is important for substrate and product release because L-serine, L-tryptophan, and aminoacrylate dissociate very slowly from the mutant alpha 2 beta 2 complex. Our ability to prepare very stable derivatives of the mutant alpha 2 beta 2 complex containing tightly bound aldimines with a substrate, a product, or a reaction intermediate provides valuable materials for ongoing x-ray crystallographic investigations and future kinetic analyses of the allosteric activation of the alpha subunit by beta subunit ligands.
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Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Nagata
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McPhie
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Miles
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D002384:Catalysis; D002942:Circular Dichroism; D007097:Imines / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D008239:Lysine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology; D013057:Spectrum Analysis; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007097:Imines; D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase; D008239:Lysine