title: Proton transfers in the beta-reaction catalyzed by tryptophan synthase.
abstract: Reactions catalyzed by the beta-subunits of the tryptophan synthase alpha(2)beta(2) complex involve multiple covalent transformations facilitated by proton transfers between the coenzyme, the reacting substrates, and acid-base catalytic groups of the enzyme. However, the UV/Vis absorbance spectra of covalent intermediates formed between the pyridoxal 5'-phosphate coenzyme (PLP) and the reacting substrate are remarkably pH-independent. Furthermore, the alpha-aminoacrylate Schiff base intermediate, E(A-A), formed between L-Ser and enzyme-bound PLP has an unusual spectrum with lambda(max) = 350 nm and a shoulder extending to greater than 500 nm. Other PLP enzymes that form E(A-A) species exhibit intense bands with lambda(max) approximately 460-470 nm. To further investigate this unusual tryptophan synthase E(A-A) species, these studies examine the kinetics of H(+) release in the reaction of L-Ser with the enzyme using rapid kinetics and the H(+) indicator phenol red in solutions weakly buffered by substrate L-serine. This work establishes that the reaction of L-Ser with tryptophan synthase gives an H(+) release when the external aldimine of L-Ser, E(Aex(1)), is converted to E(A-A). This same H(+) release occurs in the reaction of L-Ser plus the indole analogue, aniline, in a step that is rate-determining for the appearance of E(Q)(Aniline). We propose that the kinetic and spectroscopic properties of the L-Ser reaction with tryptophan synthase reflect a mechanism wherein the kinetically detected proton release arises from conversion of an E(Aex(1)) species protonated at the Schiff base nitrogen to an E(A-A) species with a neutral Schiff base nitrogen. The mechanistic and conformational implications of this transformation are discussed.
authors.affiliation: Department of Biochemistry, University of California, Riverside, CA 92521, USA.
authors.forename: Oscar
authors.initials: O
authors.lastname: Hur
authors.forename: Dimitri
authors.initials: D
authors.lastname: Niks
authors.forename: Patricia
authors.initials: P
authors.lastname: Casino
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D002021:Buffers; D002384:Catalysis; D002586:Cesium / Q000737:chemistry; D010637:Phenolsulfonphthalein / Q000737:chemistry; D011522:Protons; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000737:chemistry; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D002021:Buffers; D011522:Protons; D012996:Solutions; D002586:Cesium; D012694:Serine; D012964:Sodium; D014367:Tryptophan Synthase; D010637:Phenolsulfonphthalein; C023650:aniline