Proton Transfers in the β-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.

Tryptophan Synthase: Structure and Function of the Monovalent Cation Site

“…Solution studies and x-ray structures have shown that the MVC-bound internal and external aldimine intermediates have the open conformation, while the α-aminoacrylate and quinonoid intermediates have the closed conformation (3, 5, 15, 16, 18–22). The reaction of L-Ser with the MVC-bound and -free forms of α 2 β 2 results in the formation of equilibrating mixtures dominated by E(Aex 1 ), λ max = 424 nm, and E(A-A), λ max = 350 and 460 nm (9, 10, 11, 13, 14, 31–33). Figure 2A compares the static spectra for the reaction of L-Ser in the presence or absence of Na + , Cs + , or NH 4 + .…”

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

“…The diffraction data collected for these crystals were processed and scaled using HKL2000 (14). Both crystals belong to the space group P3 2 21. The details and statistics of the data collection are summarized in Table 4.…”

pH Dependence of Tryptophan Synthase Catalytic Mechanism

“…(v) The external aldimine accumulation is favored at high pH both in solution and in the crystalline state, whereas the ␣-aminoacrylate is stabilized at low pH (32)(33)(34). (vi) The conversion of the external aldimine to the aminoacrylate is accompanied by the release of a proton originating from the Schiff base nitrogen (24). Surprisingly, no studies have addressed the pH dependence of the catalytic rates on the S. typhimurium enzyme, the form for which the three-dimensional structure of the internal aldimine (12,15,35), the external aldimine (35,36), and the ␣-aminoacrylate (12) has been determined.…”

“…This key catalytic intermediate predominantly absorbs at 350 -360 nm extending with broad bands at 470 nm. These bands have been attributed to either the enolimine and ketoenamine tau-tomers, respectively (23), or to an unprotonated and protonated aminoacrylate Schiff base, respectively (24). In tyrosine phenol-lyase and tryptophan indole-lyase, the predominant form of the ␣-aminoacrylate absorbs at 350 nm (25), whereas in Oacetylserine sulfhydrylase, the predominant form absorbs at 470 nm (26).…”