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`
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`
`Biochemistry
`
`0 Copyright 1996 by the American Chemical Society
`
`Volume 35, Number 1
`
`January 9, 1996
`
`Accelerated Publications
`
`Direct Measurement of the Aspartic Acid 26 pKa for Reduced Escherichia coli
`Thioredoxin by 13C NMR t
`Mei-Fen Jengt and H. Jane Dyson*
`The Scripps Research Ins titute, 10666 North Torrey Pines Road, La Jolla, California 92037
`
`Received October 6, 1995; Revised Manuscript Received November 8, 1995®
`
`ABSTRACT: Because of interference from the pH-dependent behavior of nearby groups in the active site
`of Escherichia coli thioredoxin, the pKa of the buried carboxyl group of the aspartic acid at position 26
`has been difficult to quantitate. We repo1t a direct 1neasure1nent of this pK3 using an NMR 1nethod
`utilizing the conelation between the cPH proton resonances and the 13CO of the titrating carboxyl group.
`The experiments show unequivocally that the pKa is 7.3-7.5, rather than the value of9 or greater recently
`proposed by Wilson, N. A., et al. [(1995) Biochemistry 34, 8931-8939]. The assignment of the titrating
`resonances to Asp 26 is unambiguous: the values of the cPH chemical shifts conespond exactly to those
`of Asp 26, and their titration in the pH range 5. 7-10.0 is the same as that obse1ved previously for the
`proton resonances alone. In addition, the chemical shift of the carboxyl 13C resonance at pH 5.7 is upfield
`of those of the other carboxyl and carboxamide resonances, diagnostic for a protonated carboxyl group.
`The resonances assigned to Asp 26 are the only ones that titrate in the pH range 5.7-10.5. None of the
`other aspai1ate and glutamate residues in the molecule ai·e titrated in this pH range, consistent with their
`positions on the smface of the molecule. The pK. measmed for Asp 26 in reduced thioredoxin is identical
`within experimental e1rnr to that measm·ed in the oxidized fonn of the protein. This is significant for the
`reductive mechanism of thioredoxin: the bmied salt b1idged/hydrogen-bonded side chains of Asp 26 and
`Lys 57 are likely to contribute to the facility of the reaction by providing a convenient source ai1d sink
`for protons in the hydrophobic enviromnent of the complex between thioredoxin and its substrates.
`
`A number of studies have recently adch-essed the issue of
`the pK. of tl1e buried aspartic acid in Escherichia coli
`thioredoxin. This residue is highly conse1v ed among all
`th.ioredoxins (Eklund et al., 1991) and appears to be
`completely buried in both proka1yotic (Katti et al., 1990;
`Jeng et al., 1994) and eukaiyotic (Qin et al., 1994) tlJ.io(cid:173)
`redoxins. The E-amino group of a lysine residue at position
`57 is also buried in close proximity to Asp 26 and takes
`pai1 in a loose (Jeng et al. , 1994) or water-mediated (Katti
`
`t This work was supported by Grant GM43238 from the National
`Institutes of Health.
`• To whom correspondence should be addressed.
`! Present address: Graduate Institute of Cell and Molecular Biology,
`Taipei Medical College, Taipei, Taiwan.
`® AbstractpublishedinAdvanceACS AbstracL<, December 15, 1995.
`
`et al., 1991) hydrogen-bonding interaction. TlJ.is residue is
`highly conse1ved only among prokaryotes, but its function
`is apparently duplicated in eukaryotic th.ioredoxins by the
`presence of a lysine residue at position 39 (Eklund et al.,
`1991). The pK. of the buried aspai1ate residue has been
`detennined by a number of methods for the oxidized form
`of thioredoxin (Trx-S2) (Dyson et al., 1991; Langsetmo et
`al., 1991a) to be considerably shifted from "normal"
`values: to 7.5 instead of ~4.0. The pK. detemJ.ination for
`the reduced f01m of the protein [Trx-(SH)2] is complicated
`by tl1e effect of the titration of the nearby tlJ.iol groups of
`the active site cysteine residues, Cys 32 and Cys 35, but
`was infetTed from NMR studies to be also in the vicinity of
`7 (Dyson et al., 1991 ). TI1e relationship of these three groups
`is shown in Figure 1.
`
`0006-2960/96/0435-1$12.00/0
`
`© 1996 American Chemical Society
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`2 Biochemistry, Vol. 35, No. 1, 1996
`
`Accelerated Publications
`
`FIGURE 1: Portion of one of the solution structures of Trx-(SH)2 (Jeng et al., 1994) showing the spatial relationship between the two
`cysteines and the buried Asp 26 and Lys 57 side chains.
`
`The unusual pH dependence of the two active site thiols
`in reduced thioredoxin is crucial for the reductive mechanism.
`Reduction of a substrate disulfide by thioredoxin involves a
`two-electron, two-proton reduction. pH control at the active
`site is therefore important to the control of the rate of
`reduction by thioredoxin. We have recently site-specifically
`determined the pKas of the two active site thiols in Trx-(SH)2
`using direct NMR methods to be 7.5 and 9.5 (Jeng et al.,
`1995). The 13C resonance of the atom attached to the titrating
`group should primarily show the influence only of the
`directly bonded titrating group. Surprisingly, the C(cid:226) reso-
`nances of the two cysteine residues show a double titration.
`We have interpreted this as evidence of a shared proton
`between the sulfur atoms of the two cysteines at neutral pH,
`consistent with the otherwise unexplained shifted pKa of the
`solvent-exposed Cys 32 thiol.
`At the same time, another group was working on the same
`system (Wilson et al., 1995). Their 13C-1H data for the
`cysteine thiols of Trx-(SH)2 appear identical to our own, but
`a very different interpretation was placed on them. The
`higher of the two pKas observed in the 13C(cid:226)-1H(cid:226) titration,
`at 9.5, was attributed to the buried Asp 26 carboxyl group
`on the basis of a comparison of the behavior of a mutant in
`which Asp 26 had been replaced by alanine (D26A). In this
`paper we present a direct measurement of the Asp 26 pKa,
`using a modified two-dimensional H(CA)CO NMR experi-
`ment to estimate directly the 13C chemical shift of the
`carboxyl carbon of Asp 26. These measurements establish
`unequivocally that the pKa of Asp 26 is between 7.3 and
`7.5, rather than 9, as inferred by Wilson et al. (1995). This
`has profound implications for the mechanism of pH control
`in the reductive and oxidative reactions of thioredoxin.
`
`MATERIALS AND METHODS
`
`Reduced thioredoxin uniformly labeled with 15N and 13C
`was prepared as previously described (Chandrasekhar et al.,
`1991, 1994), utilizing an algal homogenate (Martek Co.) as
`a basis for an enriched medium. Purification of thioredoxin
`and preparation of D2O samples of the reduced protein using
`dithiothreitol were performed as previously described (Dyson
`et al., 1989). The pH of the sample was varied between 5.7
`
`and 10.6 by the addition of small aliquots of 0.1 M NaOD
`or DCl in D2O. pH values quoted are meter readings
`uncorrected for the deuterium isotope effect.
`NMR experiments were carried out at 308 K on a Bruker
`spectrometer operating at 500 MHz for protons. The
`behavior of the 13CO of the carboxyl and carboxamide groups
`in the protein as a function of pH was monitored using a
`two-dimensional H(CA)CO experiment (Kay et al., 1990),
`modified to optimize the detection of the coupling between
`the 13CO of a carboxyl or carboxamide and the adjacent
`13C(cid:226)H or 13CγH (Yamazaki et al., 1993; Oda et al., 1994).
`Spectra were referenced in ω2 to the pH-independent back-
`bone 13CO-1H cross peaks of Gly 84 at 4.38 ppm and Ala
`108 at 4.09 ppm (Dyson et al., 1989) and indirectly in ω1
`(Wishart et al., 1995). Spectral widths were 6250 Hz with
`2048 complex points in ω2 and 6250 Hz with 128 complex
`points in ω1. Quadrature detection was achieved in ω1 by a
`combined States-TPPI method. Spectra were Fourier
`transformed using Gaussian and exponential window func-
`tions on a Sun workstation using the FTNMR software of
`Dennis Hare.
`Chemical shift values as a function of pH for Asp 26 were
`analyzed using the program Templegraph (Mihalisin As-
`sociates) in terms of a single titration curve of the form
`(Dyson et al., 1991):
`
`δ ) δHA
`
`- ((δHA
`
`- δA)/[1 + 10n(pKa-pH)])
`
`where δ is the observed chemical shift at a given pH, δHA
`and δA are the chemical shifts for the various protonated
`forms of the protein, n is the number of protons transferred,
`and Ka is the acid ionization constant.
`
`RESULTS AND DISCUSSION
`
`A modified H(CA)CO spectrum of Trx-(SH)2 at pH 8.5
`is shown in Figure 2. All of the cross peaks can be identified
`either with the backbone 13CO-HR correlations of glycine
`and other residues whose 13CR frequency is sufficiently low
`to be excited in the experiment or with the side-chain
`13CO-H(cid:226) correlations of
`the aspartate and asparagine
`
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`
`Biochemistry, Vol. 35, No. 1, 1996 3
`
`G71 ., G97
`
`N83
`
`@)
`
`6 q
`
`0
`
`D26
`
`~
`
`~
`
`~ (cid:173)
`
`© L
`N63 :c::;
`-
`
`V::::::e
`N106
`
`D15
`~
`
`172
`
`174
`
`176
`
`17813cc
`
`180 ppm
`
`182
`
`184
`
`186
`
`4.5
`
`4.0
`
`2.5
`
`2.0
`
`3.0
`3.5
`1H ppm
`FIGURE 2: A 500 MHz modified two-dimensional H(CA)CO
`spectrum of reduced thioredoxin at pH 8.52, showing the cross
`peaks for the glycine CRH-CO (backbone) and the side-chain
`C(cid:226)H-CO and CγH-CO connectivities for the Asp, Glu, Asn, and
`Gln residues. Cross peaks are also visible for other residues for
`which (like glycine) the CR resonance is at a sufficiently low
`frequency to be excited during the pulse sequence, which has
`otherwise been optimized for the C(cid:226) and Cγ of the side chains.
`
`Table 1:
`
`13CO Assignments for Trx-(SH)2 at pH 5.92, 308 Ka
`CRH
`13CO
`Gly
`Gly 21
`3.99, 3.99
`174.7
`Gly 33
`3.98, 4.31
`176 1
`Gly 51
`4.40, 3.74
`176 2
`Gly 65
`3.98, 3.67
`178.7
`Gly 71
`3.82, 3.67
`176 3
`Gly 74
`4.20, 3.74
`172.4
`Gly 84
`4.38, 3.75
`175 1
`Gly 92
`3.59, 4.38
`173.6
`Gly 97
`3.76, 3.92
`179 1
`C(cid:226)H
`2.27, 2.18
`2.89, 2.23
`2.65, 2.21
`2.72, 2.78
`2.85, 3.11
`2.23, 1.88
`CγH
`13CO
`Glu/Gln
`185.2
`2.62, 2.68
`Glu 44
`184.6
`2.44, 2.14
`Glu 48
`186.6
`2 53, 2.50
`Gln 50
`186.6
`2 28, 2.47
`Gln 62
`186.6
`2.45, 2.45
`Gln 98
`a Assignments are presented only for those resonances that could
`be unambiguously identified from the 2D H(CA)CO spectrum.
`
`Asp/Asn
`Asp 15
`Asp 26
`Asn 59
`Asn 63
`Asn 83
`Asn 106
`
`13CO
`180.3
`173.7
`177.5
`179.1
`180.3
`178.1
`
`residues and the 13CO-Hγ correlations of the glutamic acid
`and glutamine residues.
`The 13CO chemical shifts for resonances that could be
`unambiguously assigned from the H(CA)CO spectrum are
`shown in Table 1. The cross peaks for the majority of the
`11 aspartate residues are heavily overlapped in the region
`(1H ) 2.6-2.9 ppm and 13C ) 180-182 ppm). The cross
`peaks due to Asp 26 are readily identifiable, since the C(cid:226)H
`resonances are distinctive (Dyson et al., 1989), and the
`13CO resonance is upfield-shifted, as expected for a proto-
`nated carboxyl carbon (Oda et al., 1994). However, the cross
`peaks are of lower intensity than those for the other carboxyl
`groups of the molecule, presumably reflecting the unique
`position of the Asp 26 side chain, deeply buried in the
`hydrophobic cavity behind the active site (Jeng et al., 1994).
`
`pH
`
`5.92
`
`6.49
`
`7.00
`
`7.29
`
`7.61
`
`0
`i-\-_-_-_-_-_-.:._r_-_-_-_-_-.:..•_-_-_-_-_-.:._•_-_-_-_-_-.:._.,.._-_~f 174.1
`~ 173.5
`
`~ 173.6
`
`~=====i:::::::::i:::::::::i:::::::::i::::::::'.~ 174.0
`
`~ 174.2
`
`a
`
`1-;-:::::!::::::::i::::::r::::::!:::!_t 174.7
`e F174.5
`~
`~=====i::::=====i::::=====i::::====:;;:r:=~~ 175.0
`t174.9
`lft,,,_
`D
`U F
`~=====i::::=====i::::=====i::::=====i::::==,.L 175.4 ppm
`
`13co
`
`8.521'
`
`8.08 It, ====::!o=====!:====::!====~:::o!:=~[:::
`0
`0 ~175.3
`·t=====:::=====:::=====:::=====:::=~ .... f 175.8
`0
`0 ~ 175.5
`9.021'
`9.71 I o
`,
`
`~1H~
`
`~ 175.7
`
`r· - - - - - . - - - - . - - -~ __ &_o~__Jf 116.2
`3.0
`2.8
`2.6
`2.4
`2.2
`1H~ ppm
`FIGURE 3: Portions of the 500 MHz modified two-dimensional
`H(CA)CO spectrum of reduced thioredoxin at a number of pH
`values between 5.92 and 9.71, showing the cross peaks assigned
`to the Asp 26 C(cid:226)H-13CO connectivities. These spectra are in
`general plotted at a lower contour level than that in Figure 1.
`
`All of the other carboxyl side chains and many of the
`carboxamides (Asn and Gln) are present on the surface of
`the molecule, where they are in many cases in free rotation
`even at the CR-C(cid:226) bond (Chandrasekhar et al., 1994; Jeng
`et al., 1994). This would give rise to relatively narrow line
`widths for the carboxyl carbons. For Asp 26, however, the
`side chain is fixed in the interior of the molecule, so the
`correlation time is strictly controlled by the overall tumbling
`of the molecule. It is significant that the Asp 26 cross peaks
`have a tendency to disappear in older samples and at the
`extremes of the pH range, where a small amount of
`aggregation (at pH < 6) or unfolding (at pH > 9.5) is more
`likely to occur. The concomitant
`increase in average
`to broaden the 13CO
`correlation time is then sufficient
`resonance beyond detection. A similar lowering of the cross-
`peak intensity is observed for those carboxamide residues
`that the solution structure of Trx-(SH)2 indicates are buried,
`for example, Asn 59 and Gln 98.
`The cross peaks corresponding to the Asp 26 C(cid:226)H-13CO
`are shown in Figure 3 for all pHs except 5.7 and 10.0, plotted
`at a lower contour level than for Figure 2. The pH-dependent
`behavior of the 13CO and 1H(cid:226) resonances is shown in Figure
`4 and the pKas are shown in Table 2. The magnitude of the
`shift in the 13C chemical shift, 2.2 ppm, is comparable to
`those observed for the titrations of aspartates in ribonuclease
`H1 (Oda et al., 1994).
`It is immediately obvious that the
`pKa for the transition in chemical shift between pH 5.7 and
`pH 10 is quite similar for the 13CO chemical shifts and for
`those of the H(cid:226)2 resonances [stereospecific assignments
`
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`4 Biochemishy, Vol. 35, No. 1, 1996
`
`Accelerated Publications
`
`i3co
`
`•
`
`•
`
`177
`
`176
`
`175
`
`174
`
`173
`2.90 0
`•
`
`2.80
`
`E
`a.
`-9;
`;:::
`i:
`(J)
`
`0
`
`0
`
`of a titration at pK. 2'.: 9 as suggested by Wilson et al. (1995).
`A mnnber of attempts have been made to localize tile pK.s
`of the titrating groups in the active site of reduced thio(cid:173)
`redoxin. Early cheniical modification studies indicated that
`the pK. of one of the cysteine thiols in reduced thioredoxin
`was ~6.8 and the other ~9 (K.allis & Holmgren, 1980). A
`later NMR study (Dyson et al., 1991) interpreted a large
`volume of 1H titration data in tenns of only two pK.s in the
`active site region, although it was obvious that three titrating
`groups (Asp 26, Cys 32, and Cys 35) were present. This
`approach was justified by the inference that two of the pK.s
`(Asp 26 and Cys 32) appeared to have very similar pK.s, in
`the vicinity of 7. A higher pK. of 8.4 was foru1d for Cy s
`35, closer to nonnal values for cysteine pK.s in proteins.
`However, tl1e pH range over which these measurements were
`taken was smaller, at least at the high-pH range, than later
`studies: it appears that the pK. obtained for Cys 35 was
`ru1derdetennined. Later work using specifically (Wilson et
`al., 1995) and seniispecifically labeled thioredoxin (Jeng et
`al., 1995) indicated that the higher pK. in fact more closely
`appruadu::s 9.5, as originally fuuml u y Kallis aml Hulmgn::u
`(1981) . A Raman study (Li et al., 1993) gave pK.s of 7.1
`and 7.9 for the two cysteine thiols, wiili the behavior of the
`complex Raman SH stretching band indicating that the two
`cysteine pK.s were below pH 8.2. The same work indicated
`that all of the carboxyl groups in the molecule were
`completely deprotonated below pH 8.
`TI1e question of inte1pretation of these data hinges on the
`identification of the titrations of tlrree groups, Asp 26, Cys
`32, and Cys 35. The 13C NMR indicates two pK.s for the
`cysteines of 7.5 and 9.5 (Jeng et al., 1995), while the pK. of
`Asp 26 is shown by the present work to be 7.5. The sin1plest
`explanation is that of Jeng et al. (1995): the two cysteine
`pK.s are 7.5 and 9.5 and the Asp 26 pK. is also 7.5. By
`contrast, tl1e interpretation of Wilson et al. (1995) is more
`complex. The higher of the two pK.s seen in ilie 13C
`experiments on tile cysteines is ascribed to ilie titration of
`Asp 26. Even without the direct evidence presented in tliis
`paper on the Asp 26 pK., several facts argue against this
`inte1pretation. First, while proton cheniical sliifts can be
`influenced by a number of titrating groups (Dyson et al.,
`1991 ), tl1e influence of titrating groups otl1er than the
`i1mnediately adjacent one on the 13C chemical shift of the
`adjacent carbon atom should be small: the major influence
`on the nCJl carbon cheniical shifts of the cysteines should
`therefore be the titrations of the cysteine thiols tl1emselves.
`Second, a significantly greater change in 13Cfl cheniical shift
`is seen for the pK. 9.5 transition for Cys 32 than for Cys 35,
`exactly the opposite of what would be expected from the
`relative proxiniity of the two cysteines to ilie Asp 26 side
`chain. Tiiird, ilie stmctrues ofTrx-(SH)2 and Trx-S2 are the
`same (Jeng et al., 1994): only the most subtle changes in
`dynaniics (Stone et al., 1993) and hydrogen exchange (Jeng
`& Dyson, 1995) reveal any differences between the two
`f01ms at all. It is therefore at least plausible that the pH(cid:173)
`dependent behavior of Asp 26 should be the same in ilie
`two fonns of the protein, even though the charge balance in
`the active site is more negative at high pH in Trx-(SH)2 than
`in Trx-S2. In addition, the same differences in dynaniics
`and hydrogen exchange indicate that the backbone mobility
`in the region of tl1e active site is actually sigiiificantly greater
`in Trx-(SH)2: we would therefore expect the Asp 26 pK., if
`different from that ofTrx-82, to be removed toward the lower
`
`~ 2 .70
`.E
`.c u
`
`Q)
`
`2.60
`2.30
`
`2.20
`
`1H~3
`
`•
`
`0
`
`0
`
`0
`
`0
`
`0 00
`
`0 •
`1H~2
`2.10-- ~ - - - - -~ - -- -~
`5.5
`6.5
`7.5
`10.5
`8.5
`9.5
`
`pH
`FIGURE 4: Plot of the chemical shift of the resonances shown in
`Figtll'e 2 as a ftmction of pH. Solid lines are ctu-ves fitted to the
`data (e ) using the method of least squru·es to these points. Also
`included are data points obtained previously from 1H NMR spectra
`(0) (Dyson et al., 1991). The points at the high-pH extremum
`appeat· to be influenced by another pH transition and were not
`included in the fit.
`
`Table 2: Measured pK, Values for the Asp 26 Carboxyl Group
`
`atom
`Asp 26 13C
`Asp26 1IJ/32
`
`M(ppm)
`pK,
`source
`this work
`2.2
`7.3
`this work
`(78Y,
`(003Y,
`7.3
`Dyson et al., 1991
`0.04
`this work
`0.21
`7.5
`Dyson et al., 1991
`0.22
`7.3
`• The pK, value obtained for Asp 26 1Hf32 is not well determined
`due to the small size of the chemical shift change with pH (,M).
`
`Asp 26 1IJ/33
`
`according to Chandrasekhar et al. ( 1994)]. The H/33
`resonance undergoes ve1y little change over this pH range.
`At pH values greater than 9 there appears to be another pH(cid:173)
`dependent process occruring. TI1e magnitude and abruptness
`of the change indicates that it is probably not related to the
`cysteine pK. at 9.5. There are two possible explanations
`for this- changes due to general tmfolding of the molecule
`or deprotonation of another nearby group such as the Lys
`57 E-amino group. Also included in Figure 4 are the data
`points from the previous 1H NMR study of reduced tliio(cid:173)
`redoxin (Dyson et al., 1991). TI1e chemical shift values at
`any given pH differ slightly between the two data sets,
`possibly due to ilie 10° temperatrue difference between the
`two sets of measurements. However, ilie pH-dependent
`behavior of the resonances in the 1H COSY spectra closely
`parallels that observed for the 13C- 1H HSQC measurements,
`further evidence that the low-intensity cross peaks observed
`in the latter expe1iment are indeed those of Asp 26. Most
`significantly, Figure 4 shows a pronoru1ced single titration
`in the 13CO for Asp 26, with a pK. of 7.3. There is no sign
`
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`
`•
`
`,os
`
`t ,.
`t ~ :· 110
`· . ~
`, •• 1 ... e,o ,. ..
`• •~ I
`I ~ f
`
`.. , ... ,,, . ~ 15N
`•• __. ...
`'
`
`OU~
`
`~
`
`115
`
`" '
`
`~
`
`,
`
`f
`
`c,I
`,
`·
`
`120
`
`ppm
`
`t
`
`125
`
`130
`
`'• •
`Wl
`.,
`
`•
`
`~
`\.1'
`
`' I
`-· , u•
`' .,.
`
`I ..,
`
`10.0
`
`..
`
`•
`90
`8 0
`1H ppm
`FIGURE 5: Superposition of the 500 MHz 15N-1H HSQC spectra
`of wild-type Trx-(SH)2 (black) at pH 6.49, 298 K, and D26A mutant
`Trx-(SH)2 (red) at pH 6.51, 298 K, showing the extent of the
`changes in the chemical shifts in the vicinity of the active site as
`a result of the mutation.
`
`7.0
`
`values more characteristic of solvent-exposed carboxyl
`groups, rather than higher by 2 units in Trx-(SH)2, as
`suggested by Wilson et al. (1993).
`The interpretation of Wilson et al. (1995) relies on two
`lines of evidence: changes in cysteine pKas in mutant
`proteins and Raman spectra that indicate that the cysteine
`pKas are 7.1 and 7.9 (Li et al., 1993). However, they ignore
`other Raman data in the same paper that indicate that the
`titrations of all of the carboxyl groups (presumably including
`that of Asp 26) are complete below pH 8.0, and the fact that
`neither of the NMR titration studies (Wilson et al., 1995;
`Jeng et al., 1995) shows any indication of a pKa of 7.9 for
`the cysteine 13C(cid:226) titrations. The major evidence cited by
`Wilson et al. (1995) in favor of a high pKa (>9) for the
`buried Asp 26 carboxyl group is a comparison of the results
`for wild-type reduced thioredoxin with those obtained for a
`mutant in which the aspartate side chain is replaced by an
`alanine (D26A). This mutant has been the subject of a large
`amount of experimental research (Langsetmo et al., 1990,
`1991a,b), and we have also undertaken extensive NMR and
`biochemical studies of this and other mutants in the active
`site region (Dyson et al., 1994; H. J. Dyson and A.
`Holmgren, manuscripts in preparation). Both the study of
`Wilson et al. (1995) and our own measurements indicate
`that the pKas of the two cysteine thiols in the mutant Trx-
`(SH)2 are between 7.5 and 8.0. We have made complete
`assignments of the 1H, 13C, and 15N NMR spectra of the
`D26A mutant using a combination of two- and three-
`dimensional homo- and heteronuclear spectra. These studies
`reveal that while the majority of the protein is relatively
`unaffected by the mutation, the structure in the active site
`region is greatly perturbed, as shown by large chemical shift
`differences between mutant and wild-type Trx-(SH)2 at sites
`far removed in the sequence from the immediate area of the
`mutation. This is illustrated in Figure 5, which shows a
`comparison of the wild-type and D26A mutant 15N HSQC
`
`Biochemistry, Vol. 35, No. 1, 1996 5
`
`spectra. A significant rearrangement of the hydrophobic
`pocket where the Asp 26 side chain resides in the wild-type
`protein is consistent with the observed increase in thermo-
`dynamic stability of the mutant protein (Langsetmo et al.,
`1991b), although the stability of the protein to pHs lower
`than 6.0 is decreased (H. J. Dyson, unpublished observa-
`tions). In view of the structural differences apparent between
`the mutant and wild-type proteins,
`it
`is not valid to
`extrapolate from the behavior of the mutant
`to make
`conclusions about the behavior of the wild-type protein: the
`pKas of the two cysteine thiols are most probably changed
`by a rearrangement of the active site structure in the mutant.
`In addition, the lowering of the pKa of Cys 35 from 9.5 in
`wild-type to 7.5-8 in the mutant would appear to be a logical
`consequence of the removal of the negatively charged Asp
`26 from its local environment.
`A change in the pKa of Asp 26 to 8.3 is observed in the
`double mutant C32S/C35S (Dyson et al., 1994). A pKa
`increase upon removal of the two potential negative charges
`on the cysteines is apparently paradoxical and was another
`of the justifications of Wilson et al. (1995) for their
`to the >9.0 titration. By this
`assignment of the pKa
`argument, the high pKa of Asp 26 in the wild-type protein
`is reduced in the mutant by the removal of the negative
`charges. Once again, extrapolation of mutant data to make
`conclusions about the wild-type protein is simplistic: other
`changes occur than a simple charge removalsthe group that
`has replaced the charged group must be considered, as well
`as the structural rearrangements caused by the mutation in
`the hydrophobic pocket where the Asp 26 carboxyl
`is
`situated.
`We have shown unequivocally that the Asp 26 pKa in wild-
`type reduced thioredoxin closely resembles that observed for
`the oxidized form of the protein. This is consistent with
`the high degree of similarity between the two structures (Jeng
`et al., 1994) and with a mechanism that includes a shared
`proton between two thiols as a means both of stabilization
`of the reactive thiolate form of the Cys 32 side chain and of
`promoting complex formation between reduced thioredoxin
`and protein substrates (Jeng et al., 1995). Both the oxidative
`and reductive reactions of thioredoxin are severely impaired
`in the D26A mutant, mainly in the slowing of reaction rates
`(A. Holmgren, H. J. Dyson, and I. Slaby, manuscript in
`preparation). The buried aspartate, with its pKa poised at
`the lower of the two cysteine thiol pKas, is clearly involved
`in control of the efficient proton transfers at the active site
`during the reactions of thioredoxin, probably by serving as
`a proton source and proton sink removed from the exterior
`solvent after the formation of the hydrophobic complex
`between thioredoxin and its substrate.
`
`ACKNOWLEDGMENT
`
`We thank Dr. Peter Wright for continuing helpful discus-
`sions and for a critical reading of the manuscript, Professor
`Arne Holmgren for providing the 13C-labeled protein, and
`Stefan Prytulla, Jian Yao, and John Chung for helpful
`discussions on the NMR experiments.
`
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