Mutation of an active site residue of tryptophan synthase (beta-serine 377) alters cofactor chemistry.
To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta-Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa approximately 7.7) from a form with a protonated internal aldimine nitrogen (lambdamax = 416 nm) to a deprotonated form (lambdamax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta2 subunit and alpha2 beta2 complex are pH-independent. The reaction of the S377D alpha2 beta2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambdamax = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.
Jhee
K H
KH
National Institutes of Health, Bethesda, Maryland 20892, USA.
Yang
L H
LH
Ahmed
S A
SA
McPhie
P
P
Rowlett
R
R
Miles
E W
EW
D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D005971:Glutamates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012545:Schiff Bases; D012694:Serine; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014367:Tryptophan Synthase / Q000737:chemistry*
D001426:Bacterial Proteins; D005971:Glutamates; D046911:Macromolecular Substances; D012545:Schiff Bases; D001224:Aspartic Acid; D012694:Serine; D011732:Pyridoxal Phosphate; D014364:Tryptophan; D014367:Tryptophan Synthase