Effects of Buried Charged Groups on Cysteine Thiol Ionization and Reactivity in Escherichia coli Thioredoxin:  Structural and Functional Characterization of Mutants of Asp 26 and Lys 57

Abstract

To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced [Trx-(SH)2] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26. A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins. A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances. Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9. The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK(a)s have been significantly shifted by the mutations. The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants. The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance m...

Conformation, stability, and active‐site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy

“…This is in agreement with the wild-type and D26A cysteine pKa assignments of Wilson et al (1995). It is also consistent with D26A data of Dyson et al (1997), although these authors propose that either both thiols titrate with pKa between 7 and 8, or one thiol titrates with pKa near 7.8 and the other with higher than their experimental pH (>IO).…”

“…the active site. A similar conclusion has been reached from fluorescence studies of D26A (Hanson, 1995;Dyson et al, 1997). Figure 4 compares the Raman SH band profiles (2,500-2,650 cm" interval) of wild-type thioredoxin and D26A, each normalized appropriately to the same intensity standard (Tuma et al, 1993).…”

Reactivity of zinc finger cysteines: Chemical modifications within labile zinc fingers in estrogen receptor

“…Cys-240 is flanked on either side by Ala-239 and Arg-241, whereas Cys-237 is sandwiched°between°Ser-236°and°Gln-238°(Figure°1).°Neigh-boring amino acids can determine thiol pK a and, thus, the chemical reactivity of an entire protein, as has been shown for the active enzymatic site of thioredoxin in which nearby Asp carboxyl and Lys amino groups influence local thiol pK a and thereby influence the rate of°thiol°disulfide°reactions°at°physiological°pH° [27]. Based on tandem-MS fragmentation experiments with mono-conjugated ZnF peptide, we observed that Cys-240 is much more reactive than Cys-237.…”

The protein disulphide-isomerase family: unravelling a string of folds

“…Cys-32 occurs near the start of an α-helix [35], is surfaceexposed and has a pK a of 7.1 [36], much lower than that of the free cysteine (pK a of 8.7 at neutral pH), rendering it highly reactive. At neutral pH the reactive sulphur atom of Cys-32 may share a hydrogen bond to the -SH hydrogen of Cys-35 [37].…”

“…At neutral pH the reactive sulphur atom of Cys-32 may share a hydrogen bond to the -SH hydrogen of Cys-35 [37]. The pK a of Cys-32 is thought to be decreased by a nearby buried partial charge on Asp-26 [36,38], which may serve as a general acid\base in thioredoxin-catalysed redox reactions [39]. The Cys-32 thiolate can make a nucleophilic attack on disulphides, generating a mixed disulphide that is then disrupted by Cys-35 to produce a reduced substrate protein.…”