Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.
In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.
Department of Biochemistry, University of California, Riverside 92521-0129.
P S
PS
Brzović
A M
AM
Kayastha
E W
EW
Miles
M F
MF
Dunn
D001224:Aspartic Acid / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D006898:Hydroxylamines / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012486:Salmonella typhimurium / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics*
D005971:Glutamates; D006898:Hydroxylamines; C057813:dihydroisotryptophan; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D014364:Tryptophan; D014367:Tryptophan Synthase; C018338:N-methylhydroxylamine