Kinetic Characterization of Channel Impaired Mutants of Tryptophan Synthase

Abstract

Tryptophan synthase, an ␣ 2 ␤ 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the ␣ subunit to the active site of the ␤ subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-Å hydrophobic tunnel which connects the ␣ and ␤ active sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling. In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements. We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (␤C170F and ␤C170W). Preliminary kinetic and structural evidence of a restricted tunnel in the ␤C170W has been provided (Schlichting, I., Yang, X. W., Miles, E. W., Kim, A. Y., and Anderson, K. S. (1994) J. Biol. Chem. 269, 26591-26593). The rapid kinetic analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant ␣␤ reaction. In addition, the ␤C170W mutant appears to be impaired in ␣␤ intersubunit communication.

Kinetic and Structural Characterization of Tunnel-Perturbing Mutants in Bradyrhizobium japonicum Proline Utilization A

“…In tryptophan synthase, substitution of βCys170 with Trp in the tunnel pathway significantly hindered passage of the indole intermediate between active sites and also impacted communication between subunits. 42 In the bifunctional enzyme dethiobiotin synthetase (DTBS)-diaminopelargonic acid aminotransferase (DAPAT-AT) from Arabidopsis , two mutations were made in a crevice on the surface connecting the two active sites. 43 The surface crevice was proposed to be a channel pathway for movement of the intermediate from DAPA-AT to DTBS.…”

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology

“…With regard to the suppression of TS allostery, we found parallels between the effect of the caging group in TS(aL58ONBY ai ) and the mutation of bC170, lining the intermolecular channel, to a bulky tryptophan (Figure 7A) [56,57]. This mutation caused conformational changes of the gating residues bY279 and bF280, and unfavorable movements of the COMM domain [58] with consequences on the allosteric communication pathway [52,59] and structural changes in the TrpB active site that impaired stabilization of bound serine.…”

“…As a result, activation of both the k cat value of TrpA [33] and the binding constant K d Ser of TrpB [58] were impaired 100-fold. Additionally, tryptophan bW170 obstructed the channel so that the rate constant for indole channeling was 5,000-fold reduced [57].…”

Biochemical characterization of aminopeptidase N2 from <i>Toxoplasma gondii</i>

“…Purified r Tg APN2, produced in E. coli , did not require the addition of metal ions for activity and the addition of low concentration of zinc ions inhibited its activity. Some Zn 2+ metalloproteases acquire zinc ions during the process of production in prokaryotic systems, and a subsequent addition of zinc ions significantly inhibits their activity [ 13 ]. We hypothesize that r Tg APN2 may also bind Zn 2+ metal ions during prokaryotic expression and purification.…”