Enzymes Harboring Unnatural Amino Acids:  Mechanistic and Structural Analysis of the Enhanced Catalytic Activity of a Glutathione Transferase Containing 5-Fluorotryptophan^,

Abstract

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.

Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

“…Similarly, in the human A1-1 GST (Alpha class), a rapid transition has been proposed involving motions of the flexible C-terminal segment that partially covers the G-site (31). Even the Mu class GST, where the active site is confined by two flexible loops, may display a similar fluctuating accessibility (32). On the whole, it appears that the very similar flexibility profiles found in Alpha, Pi, and Mu GSTs (10) are not incidental but reflect a precise evolution strategy to enhance the catalytic efficiency of the GST superfamily.…”

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies

“…Moreover, it has been shown that mutation of the Met residues in CaM to other amino acids, even to the related hydrophobic Leu residues, may impair the CaM activation of some target proteins~Zhang et al, 1994b;Chin & Means, 1996;Chin et al, 1997b;Edwards et al, 1998!. Substitution of amino acids in proteins with unnatural amino acid analogs is a relatively old approach in protein chemistry~for reviews, see Richmond, 1962;Hortin & Boime, 1983;Wilson & Hatfield, 1984!. It has seen somewhat of a revival in recent years for recent examples, see Muller et al, 1994;Randhawa et al, 1994;Thomson et al, 1994;Budisa et al, 1995Budisa et al, , 1997Budisa et al, , 1998Xiao et al, 1997;Furter, 1998;Parsons et al, 1998!. When the unnatural amino acid is incorporated to a high level into the protein, one can potentially examine its effects on the protein's properties.…”

Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue

“…It had previously been demonstrated that GST retained function (and thus could be readily purified) when 5fW was widely incorporated in place of tryptophan (27), and we anticipated that it might also retain function (and thus be amenable to purification) following 4fW incorporation. After induction in media containing either tryptophan or 4fW, GST was isolated and HPLC-ESI MS was performed on samples.…”

“…Based on previous experiments by Wong (37), we hypothesized that a relatively small number of proteins which were adversely affected by 4fW would mutate to accommodate the analogue. The fluorine-for-hydrogen substitution adds only ϳ0.15 Å in diameter, and this substitution was expected to be generally insignificant, as has previously been observed when the activities of fluorotryptophan-substituted enzymes have been examined (27). It was also possible that the fluorine substitution would alter the electronic properties of tryptophan.…”