Rota-microspeciation of serine, cysteine, and selenocysteine

J. Phys. Chem. 1991, 95, 9609-9614

9609

Rota-Microspeciation of Serine, Cysteine, and Selenocysteine BQla N ~ s z P lWei , ~ Cuo, and Dallas L.Rabenstein*

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 9, 2015 | http://pubs.acs.org Publication Date: November 1, 1991 | doi: 10.1021/j100176a102

Department of Chemistry, University of California, Riverside, California 92521 (Received: April 18, 1991; In Final Form: June 25, 1991)

Populations of the three rotamers, with respect to rotation around the C,-C, bond, have been determined from vicinal 'H-IH coupling constants for every protonation state of serine, cysteine, and selenocysteine. Rotamer populations were determined for the (COO-, NH2, OH), (COO-, NH3+,OH), and (COOH, NH3+,OH) protonation forms of serine, using a literature assignment of the two P-CH2 resonances to HA and HB of the serine CHCH2 ABX spin system. For selenocysteine and cysteine, the two @-CHIresonances were assigned to HA and HBby using both vicinal 'H-'H and I3C-'H coupling constants. Rotamer populations were determined for the (COO-, NH2, Se-), (COO-, NH3+, Se-), (COO-, NH3+,SeH), and (COOH, NH,+, SeH) forms of selenocysteine and the (COO-, NH2, S-),(COO-, NH3+,SH), and (COOH, NH3+, SH) forms of cysteine. Populations of the six rotamers of the two isomeric monoprotonated forms of cysteine, (COO-, NH2, SH) and (COO-, NH3+,S-), were estimated by considering rotamer populations for the corresponding forms of serine (COO-, NH2, OH) and selenocysteine (COO-, NH,', Se-) as models. Protonation constants were also calculated for each rotational isomer, including rota-microconstants for the basic region of cysteine where 12 rotamers coexist. It was found that the major factors governing the rotamer populations, and thus the apparent protonation constants of individual groups in rotational isomers, are intramolecular hydrogen bond formation between the hydroxyl, carboxyl, and ammonium groups for serine and repulsion between the thiolate-carboxylate and selenolate-carboxylate groups of cysteine and selenocysteine, respectively. The pH-dependent distribution of the relative concentrationsof the 15 cysteine rota-microspecies over the pH range 0-14 is presented graphically. A brief description of methodology for determination of rota-microconstants is also presented.

Introduction Most biological molecules are polybasic compounds that protonate in a stepwise fashion with specific sites protonated. If two or more protonation sites are of similar basicity, the molecules can exist as protonation isomers, e.g., the (NH3+,S-,COT) and (NH,, SH, COT)protonation isomers of the monoprotonated form of cysteine. Further, if the biological molecules are conformationally flexible, the various protonated microspecies can exist in several rotational forms, or rota-microspecies. Since both protonation and intramolecular rotation are in most cases very fast processes, protonation and rotational isomers always occur in the presence of each other and give composite analytical signals. However, because different protonation and rotational isomers can have different properties in highly specific biochemical processes, characterization of microspecies and rota-microspecies of biological molecules is an important step toward understanding physiological phenomena such as receptor binding, enzyme activity, and membrane penetration at the molecular level. The coexistence of protonation and rotational isomers and the concomitant composite analytical signals prevent the direct determination of their concentrations. However, microspeciation' and rota-micro~peciation~*~ methods have been developed for determination of the relative concentrations of microspecies and rota-microspecies and for determination of rota-microprotonation constants. Solution concentrations of microspecies and rotamicrospecies can then be calculated from the relative and total concentrations. The number of rota-microspecies and rota-microconstants increases with both the number of basic sites and the number of rotational axes in the molecule and, consequently, rota-microspeciation has been possible only for relatively small biomolecules. Rota-microspeciation is of interest not only for characterizing the distribution of biological molecules among their rotational and protonation isomers but also because it can provide information about the effect of near (gauche) and remote (trans) substituents on the basicity of protonation sites. In the present study, the rota-microspecies and rota-microconstants of the amino acids serine, cysteine, and selenocysteine have been characterized. The different acidities of the OH, SH, and SeH substituents result in distinctive acid-base properties at both the macroscopic and microscopic levels for this series of structurally analogous molecules. In the case of serine, the Permanent address: Department of Inorganic and Analytical Chemistry,

L. EBtvbs University, Pgzmgny park 1, 1 1 17 Budapest, Hungary.

carboxylate and amino groups are protonated in a well-separated twestep process4 and rotamer populations have been characterized from three-bond proton-proton or proton-carbon spinspin coupling constants.2-s-6 For cysteine, where the amino and thiolate protonations overlap, the macroscopic and microscopic equilibria have been studied by a variety of techniq~es."'~ Much less is known about the rotamer populations of the various protonated forms of cysteine,lsJ6 and no studies have been reported on the rotamer analysis of cysteine in the neutral-basic region, where 12 rota-microspecies coexist, six of which are isomeric with respect to protonation and rotation. For selenocysteine, protonation of the amino, selenolate, and carboxylate groups takes place in three well-separated steps." We report populations for the three rotamers with respect to rotation around the C,-C, bond of the (COO-, NH,, OH), (COO-, NH3+, OH), and (COOH, NH,+, O H ) forms of serine and the (COO-,NH,, Se-), (COO-,NH3+,Se-),(COO-, NH3+, SeH), and (COOH, NH3+, SeH) forms of selenocysteine. The rotamer populations were determined from three-bond IH-IH coupling constants measured at specific pD values in D 2 0 solution. Populations of the three rotamers of the (COO-, NH2, S-),(COO; NH3+,SH), and (COOH, NH3+, SH) forms of cysteine were also (1) NoszAl, B. J . Phys. Chem. 1986, 90, 6345-6349. (2) Fujiwara. S.;Ishizuka, H.; Fudano, S . Chem. Lett. 1974,1281-1284. (3)Noszil, B.; Sindor, P. Anal. Chem. 1989,61, 2631-2637. (4)Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum Press: New York, 1974;Vol. 1, Amino Acids, p 35 and references therein. ( 5 ) Hansen, P. E.;Feeney, J.; Roberts, G.C. K.J . Magn. Reson. 1975, 17, 249-261. (6)Ogura. H.;Arata, Y.; Fujiwara, S . J. Mol. Spctrosc. 1%7, 23,7645. (7)Ryklan, L. R.;Schmidt, C. L. A. Arch. Biochem. 1944,5, 89-98. (8) Benesch, R. E.; Benesch, R. J . Am. Chem. Soc. 1955,77,5877-5881. (9)Grafius, M. A.; Neilands, J. B. J. Am. Chem. Soc. 1955, 77, 3389-3390. (10) Elson, E. L.;Edsall, J. T. Biochemistry 1962,I, 1-7. (I I ) Wrathall, D. P.; Izatt, R. M.; Christensen, J. J. J . Am. Chem. Soc.

1964,86,4779-4783. (12)Coates, E.; Marsden, C. G.; Rigg, B.J. Chem. Soc., Faraday Trans. 1969, 65,3032-3036. (13)Clement, G . E.; Hartz, T.P. J . Chem. Educ. 1971,48, 395-397. (14) Reuben, D. M. E.; Bruice, T. C. J . Am. Chem. SOC.1976, 98. 114-121. (15)Walters, D.C.;Leyden, D. E. Anal. Chim. Acta 1974,72,275-283. (16)Martin, R. B.; Mathur, R. J . Am. Chem. Soc. 1965,87,1065-1070. (17)Arnold, A. P.; Tan, K.-S.; Rabenstein, D. L. Inorg. Chem. 1986,25, 2433-2437.

0022-3654/91/2095-9609$02.50/00 1991 American Chemical Society

9610 The Journal of Physical Chemistry, Vol. 95, No. 23, 1991

OH I

OH I iH2 -0OC-CH- NH;

-OOC-CH- NH2

H&OH -0OC i NH2 H

Kit

H& . o.H+ -0OC A N$

t

,I,


HO& 'OOC NH2

tN

Klg

,

9 H.&HH -0OC NH2 OH I

h

K2

NH;

-. K2t

-

,

H&H H

-ooc

NH; OH I

hN

Ss!

I

I

H H..OH HOOC H NH;

K1

/

y 2 'OOC -CH- NH2

y 2 -0OC- CH-N$

'OOC

4

NHz

-0OC

I

H

t

S' e.&H -0OC

SeH

-

y 2 'OOC-CH-NH;

SeH

K3

-

I

I

CH2 HOOC- :H-NH;

&

= H&Se H! ! I & H&eH H kHS e H

H&SZ H

tN,C

gN,C

K2

S'e

- I

I

H O . ~H H O O C ~ N ~

gN

Klh

OH

CH2 I HOOC- CH-N$

K2g

HO.&,H

-ooc A

-

Noszll et al.

NH;

,I,

O ' OC

NH;

tN

H

,I,

tN,Se

H

K1g

,I,

e-Se&H

NH2

Kzs

C

NH;

'OOC

I; N$

HOOC

tNl%,C

H K3g H HSe.&H C HSe& .-H 'OOC NH; HOOC NH;

,I,

,I,

hN#,

'N,S,C

.-- . K2h

H H.&H HOOC d H ~ ~ ; N' ,C

Figure 1. Stepwise macroscopic protonation scheme (top) and the ro-

tamers and rota-microprotonation scheme (bottom) for serine.

determined directly from three-bond 'H-'H coupling constants. Populations of the six rotamers of the two isomeric monoprotonated forms of cysteine, (COO-, NH3+, s-)and (COO-, NH2, SH), were estimated by considering rotamer populations for the corresponding deprotonated form of serine (COO-, NH2, OH) and monoprotonated form of selenocysteine (COO-, NH3+, Se-) as models. Populations are reported for nine serine, 15 cysteine, and I2 selenocysteine rota-microspecies. Rota-microprotonation constants are also reported for all rotamers of serine and cysteine in D 2 0 and H 2 0 and for selenocysteine in D20, and the pH-dependent distribution for all cysteine rota-microspecies is presented graphically.

h

hN

Figure 2. Stepwise macroscopic protonation scheme (top) and the rotamers and rota-microprotonationscheme (bottom) for selenocysteine.

:-

Theory ProtonationSchemes. The major protonation pathway of serine is shown in Figure 1. K Iand K 2 are macroscopic protonation constants. In principle, the singly protonated species (COOH, NH2, OH) is also possible; however, the basicity of the amino group is several orders of magnitude larger than that of the carboxylate group and thus the concentration of the (COOH, NH2, OH) species is negligible relative to that of (COO-, NH3+, OH). Each protonated form of serine can exist as three rotamers with respect to rotation around the C,-C, bond, as shown in Figure 1 .18J9 The labels t and g designate rotamers in which the substituent on the @-carbon(the hydroxyl group) and the bulkiest substituent on the a-carbon (the carboxyl(ate) group) are in trans and gauche positions, r e ~ p e c t i v e l y . ~ ~In~ *the * ' ~rotamer labeled h, all three carbon-bonded hydrogens are adjacent to each other, as are the three bulky groups. The subscripts (if any) indicate that the amino (N) and/or carboxylate (C) groups are protonated. The constants KI,, KZg,etc. in Figure 1 are rota-microprotonation constants for the protonation equilibria in terms of the nine rotamers of serine. The major protonation pathway for selenocysteine is shown at the top of Figure 2. Although alternative protonation pathways involving the monoprotonated species, (COO-, NH2, SeH) and ( O H ,NH2, Se-), and the diprotonated species, (COOH,NH2, SeH) and (COOH, NH3+, Se-), are possible, the relative concentrations of these species are negligible because of the relative basicities of the amino, selenolate, and carboxylate groups," and (18) Martin, R. B. J . Phys. Chem. 1979, 83, 2404-2407. (19) Espersen, W. G.; Martin, R. B.J . Phys. Chem. 1976,80, 741-745.

-0OC

'- % h

e K3 h

H&H

H.&H 'OOC NH,

H*H -0OC NH, SH

Jr gSv

I

Nq

SH h,,,

H.&H

HOOC

I NH; SH

'N,S,C

hs

Figure 3. Stepwise microscopic protonation scheme (top) and the rotamers and rota-microprotonation scheme (bottom) for cysteine.

thus these minor protonation pathways are not considered here. Also shown in Figure 2 are the protonation pathways in terms

The Journal of Physical Chemistry, Vol. 95, No. 23, 1991 9611

Rota-Microspeciation of Amino Acids

Scheme I. Y represents OH, Se-, or S-. By combining eqs 6-8, we obtain

SCHEME I

3JAX fg


1

"A

Y

(I

h

of the 12 rotamers of selenocysteine and the corresponding rota-microprotonation constants. Because the basicities of its amino and thiolate groups are similar, the protonation pathway for cysteine is more complicated, as shown at the top of Figure 3. Protonation of the amino and thiolate groups is described in terms of the microscopic protonation constants kN, kS, ksN, and kNS,where the superscripts indicate the functional group protonating in the given process and the subscript (if any) denotes a group already protonated. The macroscopic protonation constants K I and K2 for protonation of Cyszto form HCys- and protonation of HCys- to form H,Cys are composites of the microscopic constants:

K I = kN + kS

(1)

K l K 2 = kNkNS= kSksN

(2)

Also shown in Figure 3 are the protonation pathways in terms of the 15 rotamers of cysteine and the corresponding rota-microconstants. Calculation of Rotamer Populations. The rotamer mole fractions can be calculated from equilibrium considerations and three-bond spin-spin coupling constants. Taking as an example the three differently protonated g rotamers of serine, their mole fractions are defined as follows. At high pH, where serine is present as the fully deprotonated, negatively charged species (3) At neutral pH, the monoprotonated species overwhelmingly predominate and (4)

When the solution is very acidic, only the doubly protonated species are present: kN,CI fgN.C

=

[tN,CI + [gN,CI + [hN,CI

(5)

Rotamer mole fractions can be determined from vicinal 'H-IH coupling constants for the CHCH2 ABX spin systems and from vicinal I3C-IH coupling constants for the 02"CCHCH2 ABMX spin system, where M is the carboxylate carbon. Because of rapid interconversion among rotamers and the dependence of rotamer populations on the protonation state of the acidic groups, the coupling constants )JAX,j J B X ,' J A M and , 'JBMare dependent on pH. The experimental coupling constants 'JAX and 'JBXare a function of rotamer mole fractions,18-20,21 as given by eqs 6-8 for serine, selenocysteine, and cysteine at high pH: 3JAX

=hJG+ f g J T

'JBX

= A J T + /gJG + h J G f,+f8+fh=

+hJG

1

- JG

= JT - JG

(9)

Rotamer mole fractions f g andf, can be calculated from experimental values for JAX and JBX and standard values for JT and JG, and then rotamer mole fraction fh can be obtained from the relationship fh = 1 - fe-A. The generally accepted values for JG and JT are 2.4 and 13.3 Hz18 and 2.56 and 13.6 Hz;22the latter values were used in this work. For the assignment of HA and HB in Scheme I, the vicinal I3C-lH coupling is described by eqs 1 1 and 12, where M represents

=AJb+fkJb+ f h J $

(1 1)

(12) = f , J b +f g J $ +f h J b J b and J i are the 13C02group in an ABMX spin system and coupling constants for coupling between the carboxylate carbon and &protons in the gauche and trans positions, respectively. By combining eqs 8, 11, and 12, the following equations are obtained: 'JBM

Rotamer mole fractions f h andf, can be calculated from experimental values for JAM and JBM and standard values for J + and J b , and thenf, can be calculated fromf, = 1 - f -fh. Values reported for J b and J $ are 1.2 and 10.0 HzlS and 0.4 and 1 1.9 H Z ; the ~ ~former values were used here. From the above, it is apparent that, to calculate f 8 andf, with eqs 9 and 10, the two resonances for the two &CH2 protons must be assigned to H A and HB in Scheme.1, whereas the value calculated forfh is independent of the assignment. Likewise, calculation offh and f from the I3C-IH coupling constants requires that JAMand JBM %eassigned, whereas the value calculated for f, is independent of the assignment. In the absence of other information, the assignments can be made by comparison of the rotamer mole fractions calculated by using the two possible assignments of the vicinal IH-IH coupling constants with those calculated using the two passible assignments of the vicinal I'C-IH coupling constants. The correct assignments are those that give the same rotamer populations for both the IH-IH and the 13C-'H coupling constants. Determinationof Rota-Microconstants, Rota-microprotona tion constants can be determined from the macroscopic or microscopic protonation constants and the rotamer mole fractions. To illustrate, the first macroscopic protonation constant of serine can be expressed in terms of the concentrations of the rota-microspecies:

KI =

k N 1 + lhN1 (it1 + [gl + [hl)[H+l [tN1 +

(15)

The rota-microprotonation constant for the g rotamer is defined as

(6) (7) (8)

JG and JT are the standard coupling constants for coupling between protons in gauche and trans (anti) positions, respectively, with HA and HB assigned to the two @CH2 protons as indicated in (20) Abraham, R. J.; McLauchlan, K. A. Mol. Phys. 1962,5, 513-523. (21) Pachler, K. G. R. Spectrochirn. Acta 1964, 20, 581-587.

Combining eqs 3, 4, 15, and 16 leads to

Note that the average of rota-microconstants does not necessarily (22) Hansen, P. E.; Feeney, J.; Roberts, G. C. K. J . Magn. Reson. 1975,

17, 249-26 I .

9612 The Journal of Physical Chemistry, Vol. 95, No. 23, 1991 TABLE I: Constants for Three-BondSpin Coupling between the C,H and CbHi Protons of Serine, Selenocysteine, and CysteinePr compound

serine selenocysteine

cysteine

DH*

'JAY

3Jar

12.8 6.9 0.4 12.9 8.6 4.3 0.4 13.2 6.8 0.4

4.21 3.82 3.44 3.36 4.20 4.58 4.40 3.50 4.12 4.35

5.80 5.51 4.35 9.46 8.35 5.70 5.53 9.50 5.80 5.60

*In D20at 25 OC. bpH* indicates the pH meter reading in D'O solutions. eAt these pH* values, specific protonation forms predominate.


equal the macroscopic or microscopic constant. Experimental Section

Serine and cysteine were obtained from Sigma Chemical Co. and were used as received. Selenocysteine (Sigma) was reduced in situ with deuterated dithiothreitol (Merck Sharp and Dohme Inc.) to produce selenocysteine. All selenocysteine solutions were prepared in a nitrogen atmosphere in an air-tight, oxygen-free glovebox. NMR tubes were sealed before removing them from the glovebox. The solvent D 2 0 was obtained from Icon Services, Inc.; DCI and KOD were obtained from Aldrich Chemical Co. Serine and cysteine protonation macroconstants were determined in D 2 0 by potentiometric titrations at 25.0 f 0.1 O C . The ionic strength was held constant at 0.3 mol dm-3 in all the pH metric, UV, and NMR experiments, using KCI as an auxiliary electrolyte. The initial DCI and the titrant KOD concentrations were between 0.1 and 0.3 mol dm-3. Titrations were carried out by using a Mettler DV automatic buret and an Orion 701A pH meter, equipped with an Ingold combination microelectrode (6030 No. 3). The pH meter was standardized, using aqueous pH 4.008 and 9.180 buffer solutions. The pH values reported are pH meter readings, indicated by pH*. Microconstants for cysteine in D 2 0 were determined by a combination of UV spectroscopy and pH metry.8 The UV measurements were made on a Hewlett-Packard 8451A diode array spectrophotometer and the thiol group deprotonation was selectively monitored at 235 nm. 'H NMR spectra were measured at 500 MHz and 25 "C with a Varian VXR-500s N M R spectrometer, operated in the pulse-Fourier transform mode. A 90° pulse angle and a spectral range of 5000 Hz were used. The free induction decay was digitized into 30K or 34K data points. Typically 8 or 16 transients were coadded and a 15-s repetition time was used. I3C N M R spectra were measured at 125 MHz without proton decoupling. At 500 MHz, the molecules studied here show a sufficiently high ratio of chemical shift difference to coupling constant to apply first-order rules of spectral analysis.23 The values obtained by first-order analysis were then refined using a LAOCOON spin simulation and iteration computer program. The rms errors of the best-fit spectra obtained by iteration were less than 0.04 Hz. No major differences were found between the parameters obtained by first-order analysis and computer fitting of the spectra. Results and Discussion Vicinal lH-'H coupling constants were determined from IH N MR spectra measured for serine, selenocysteine, and cysteine at the pH* values reported in Table I. At these pH* values, specific protonation forms predominate. As discussed above, the two resonances for the &CH2 protons must be assigned to HA and HB to calculate rotamer mole fractions from the vicinal IH-IH coupling constants. For serine, the high-field &CH2 resonance has been assigned to the &CH2 proton labeled HB in Scheme 1. (23) GGnther, H.N M R Specrroscopy; Wiley and Sons: Chichester, 1980; p 40.

Noszll et al. The values determined for JAx and JBx for this assignment are listed in Table I and rotamer populations calculated for the specific protonation forms of serine from these values of JAxand JBx are reported in Table 11. The two vicinal IH-IH coupling constants for selenocysteine and cysteine were assigned to Ja and JBx by using both 'H-IH and 13C-'H coupling constants as described above. To illustrate, vicinal 'H-IH coupling constants of 4.35 and 5.60 Hz were determined from the * H spectrum and vicinal I3C-IH coupling constants of 2.31 and 5.76 Hz were measured from the natural abundance I3C spectrum for cysteine at pH* 0.4. Using eqs 9 and 10, rotamer mole fractions off, = 0.16,f, = 0.28, andfh = 0.56 were calculated by setting JAx = 4.35 Hz and JBx = 5.60 Hz, whilef, = 0.28,fl = 0.16, andfh = 0.56 were calculated for JAx = 5.60 Hz and JBx = 4.35 Hz. Using eqs 13 and 14, rotamer mole fractions offh = 0.52,f, = 0.13, andf, = 0.35 were calculated by taking JAM = 5.76 Hz and JBM= 2.31 Hz, whilefh = 0.13, fg = 0.52, andf, = 0.35 were calculated for JAM = 2.31 Hz and JBM 7 5.76 Hz. By comparison of the four sets of rotamer mole fractions, we conclude that JAx = 4.35 Hz, JBx = 5.60 Hz, J A M = 5.76 Hz, and J B M = 2.31 Hz (Tables I and III), and the high-field resonance of the @CH2 protons of cysteine was assigned to the proton labeled HB in Scheme I. The high-field resonance of the /3-CH2 protons of selenocysteine was also assigned to the proton labeled HB by this method. Rotamer mole fractions calculated from the experimental values for JAxand JBx are reported in Table 11. Because the standard values for JG and J , for vicinal 'H-IH coupling are known with greater certainty than Jb and J $ for vicinal I3C-IH coupling, rotamer mole fractions calculated from IH-IH coupling constants are considered more accurate and are the only mole fractions reported here. The rotamer mole fractions calculated from 13C-'H coupling constants were used only to establish the assignment of the two /3-CH2 resonances to HA and HE. There are two blank entries for serine in Table 111 because the hydroxyl group does not dissociate over the normal pH range. No populations are given for the (COO-, NH2, SeH) form of selenocysteine because protonation of the amino group is essentially complete before protonation of the selenolate begins." Rotamer populations are dimensionless proportions and are estimated to have uncertainties of f0.02-0.05. Rotamer populations could not be determined directly for the two monoprotonated forms of cysteine, (COO-, NH3+,S-)and (COO-, NH2, SH), because they coexist in solution at all pD values and consequently the observed values for 35,4X and 3JBX are weighted averages of those for the two protonation isomers. Examination of the results in Table I1 reveals that, for each of the amino acids studied here, the population of rotamer h increases with protonation, at the expense of rotamer t. For cysteine and selenocysteine, rotamer t is most abundant at high pH*, presumably to minimize unfavorable electrostatic interactions between the negatively charged carboxylate and thiolate and carboxylate and selenolate groups. In contrast, serine has only a negative carboxylate group at high pH* and rotamer h is most abundant. Protonation of the amino group of serine causes a small change in rotamer populations, whereas protonation of the carboxylate group causes a significant increase in the abundance of rotamer h, presumably because of stabilization of the (COOH, NH3+, OH) form by intramolecular hydrogen bonding between the COOH and O H groups. In contrast, the data show that, for cysteine and selenocysteine, protonation of the carboxylate group causes no change in their rotamer populations. Perhaps the most interesting feature of the results in Table I1 is that the rotamer populations of cysteine and selenocysteine are identical for the least and most protonated forms, despite the different atomic radii of sulfur and selenium. The rotamer populations are also similar for all three compounds at the (COO-, NH3+,YH) state of protonation. On the basis of these similarities, we estimate rotamer populations for the two isomeric monoprotonated forms of cysteine by assuming that the rotamer pop ulations of the (COO-,NH2, YH) forms of serine and cysteine will be similar and that the rotamer populations of the (Coo-,

The Journal of Physical Chemistry, Vol. 95, No. 23, 1991 9613

Rota-Microspeciation of Amino Acids

TABLE 11: Rotamer Powlations of Serine, Cysteine. and Selenocysteine at Different Stages of Protonation in D@"-d

protonation state of groups COO-, NH2, YCOO-, NH3+, YCOO-, NH2, YH COO-, NH3+, YH COOH, NH3+, Y H

serine PH*

t

cysteine g

h

pH*

t 0.63 0.52

13.2 12.8 6.9 0.4

0.15 0.1 1 0.08

0.29 0.27 0.16

0.55 0.61 0.76

6.8 0.4

g

h 0.29 0.33

0.09

0.29

0.15 0.15

0.29 0.28

0.14 0.16

0.55 0.57 0.56

pH*

selenocysteine t g

12.9 8.6

0.63 0.52

0.07 0.15

0.30 0.33

4.3 0.4

0.28 0.27

0.18 0.17

0.54 0.56

h

"Rotamer populations were calculated from the values for JAx and J B X in Table I by using eqs 8-10, except for the values in italics for cysteine. For the (COO-, NH3+,S-) protonation form of cysteine, JAxand J B X are taken to be similar to JAx and JBx for (COO-, NH3+,Se-) and those for the (COO-, NH2, SH) form are assumed to be the same as those for (COO-, NH2, OH). bThe pH* values are the meter readings at which the NMR measurements were made. 'Y represents oxygen, sulfur, and selenium in serine, cysteine, and selenocysteine, respectively. Blank entries are for protonation forms, which have negligible concentrations at all pH values.


TABLE III: Three-Bond Carboxylate Carboid8H2 Coupling Constants for Selenocysteine and Cysteine-

compound selenocysteine cysteine

PH*

JAM

11.4 8.8 12.8 6.4 0.4

3.17 4.03 2.69 5.41 5.76

TABLE I V Macroscopic, Microscopic, and Rota-Microprotonation Constants of Serine, Setenocysteine, and Cysteine-

macro- or microscopic protonation constd symbol value

3JBM

1.95 1.90 1.88 1.67 2.3 1

compound serine

sol-

vent D20

" In D20at 25 OC. bpH* indicates pH meter reading in D 2 0 solution.

Rota-microspeciation of serine, cysteine, and selenocysteine

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