Structure and Function of the Tryptophan Synthase α2β2 Complex

Abstract

To probe the structural and functional roles of activesite residues in the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium, we have determined the effects of mutation of The tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.2.1.20) is a useful system for investigating relationships between protein structure and function, allosteric communication between subunits in a multienzyme complex, and substrate channeling (for reviews, see Refs. 1-5). The ␤ subunit 1 is the prototypic member of a family of pyridoxal phosphate (PLP) 2 -dependent enzymes that have distantly related sequences and that catalyze ␤-replacement and ␤-elimination reactions (6 -8). The crystal structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (9) suggested the probable fold of other members of this family. Recent structures of biosynthetic threonine deaminase from Escherichia coli (10) and of O-acetylserine sulfhydrylase from S. typhimurium (11) confirm that these enzymes have the same fold as the tryptophan synthase ␤ subunit.

pH Dependence of Tryptophan Synthase Catalytic Mechanism

“…7). However, ␤His-86 was proposed to be involved in the interaction with the PLP phosphate group and, possibly, in the lowering of the pK a of ␤Lys-87 (32). There are also ionizable groups on PLP, but spectral evidence indicates a pH independence for their ionization state during the catalytic cycle (28,33,34).…”

“…(iii) The steady-state parameters of Salmonella typhimurium wild type TS are pH-dependent, indicating the presence of at least three ionizable residues with pK a1 ϭ 6.5, pK a2 ϭ 7.25, and pK a3 ϭ 8.2-9 (31). (iv) The pH dependence of ␤-replacement and ␤-elimination activity was found to be significantly altered in the ␤His-86 3 Leu mutant (32). (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).…”

“…(iv) The pH dependence of ␤-replacement and ␤-elimination activity was found to be significantly altered in the ␤His-86 3 Leu mutant (32). (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).…”

Large conformational changes in the Escherichia coli tryptophan synthase β2 subunit upon pyridoxal 5′‐phosphate binding

“…The His86 side chain, which is located in the peak 2 region next to the Lys87 of the PLP‐binding residue, shifted in location near the PLP upon PLP binding. The H86L mutant of the St α 2 β 2 β subunit exhibited a PLP‐binding ability that was reduced by approximately 20‐fold [40], indicating the importance of His86 in PLP binding. The conformational changes in His86 upon PLP binding may correlate directly with interactions between the N and C domains, leading to the closure of the holo‐β 2 subunit.…”

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants

“…Tryptophan synthase, catalyzing the final step of tryptophan biosynthesis, is one of the most rigorously documented examples of an enzyme complex [3][4][5][6]. It consists of an D subunit, which cleaves indoleglycerol phosphate to indole and glyceraldehyde 3-phosphate, and a E subunit, which condenses indole and L-serine to yield L-tryptophan.…”