Effect of pH and monovalent cations on the formation of quinonoid intermediates of the tryptophan synthase alpha(2)beta(2) complex in solution and in the crystal.
Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate-dependent enzymes. Whereas the structures of other pyridoxal 5'-phosphate-bound intermediates have been determined, the structure of a quinonoid species has not yet been reported. Here, we investigate factors controlling the accumulation and stability of quinonoids formed at the beta-active site of tryptophan synthase both in solution and the crystal. The quinonoids were obtained by reacting the alpha-aminoacrylate Schiff base with different nucleophiles, focusing mainly on the substrate analogs indoline and beta-mercaptoethanol. In solution, both monovalent cations (Cs(+) or Na(+)) and alkaline pH increase the apparent affinity of indoline and favor accumulation of the indoline quinonoid. A similar pH dependence is observed when beta-mercaptoethanol is used. As indoline and beta-mercaptoethanol exhibit very distinct ionization properties, this finding suggests that nucleophile binding and quinonoid stability are controlled by some ionizable protein residue(s). In the crystal, alkaline pH favors formation of the indoline quinonoid as in solution, but the effect of cations is markedly different. In the absence of monovalent metal ions the quinonoid species accumulates substantially, whereas in the presence of sodium ions the accumulation is modest, unless alpha-subunit ligands are also present. Alpha-subunit ligands not only favor the formation of the intermediate, but also reduce significantly its decay rate. These findings define experimental conditions suitable for the stabilization of the quinonoid species in the crystal, a critical prerequisite for the determination of the three-dimensional structure of this intermediate.
Mozzarelli
A
A
Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy. biochim@ipruniv.cce.unipr.it
Peracchi
A
A
Rovegno
B
B
Dalè
G
G
Rossi
G L
GL
Dunn
M F
MF
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011809:Quinones / Q000737:chemistry; D012964:Sodium / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000737:chemistry*; D019824:beta-MSH / Q000737:chemistry
D011809:Quinones; D019824:beta-MSH; C015102:dehydroalanine; D012964:Sodium; D014367:Tryptophan Synthase; D000409:Alanine