The Physical Biochemistry of Thiol Ionization

1. The Physical Biochemistry of Thiol Ionization Mark Wilson May 21st, 2009

2. Cysteine pKa values must be depressed for thiolate formation pKa~8-9 Henderson-Hasselbalch equation At pH=7.4, about 7% of thiol is ionized

3. Electrostatic thiolate stabilization Positive charges (e.g. lysine, arginine, metals) will electrostatically stabilize thiolate anion formation Coulomb potential energy r is distance, q is charge; always pairwise additive The dielectric constant  is a term that quantifies the bulk polarizability of the medium =80 for water =30 for methanol =1.9 for hexane =1.0 for air (and vacuum) Structural microenvironment of cysteine has a profound impact on electrostatics

4. Hydrogen bonding to the thiolate Hydrogen bond donation to cysteine commonly lowers pKa

5. The α-helix macrodipole The peptide dipole C; δ- http://web.chemistry.gatech.edu/~williams/bCourse_Information/6521/ The sum of the peptide dipoles leads to partial positive charge at the N-terminus of the helix Only the first turn contributes significantly to pKa depression N; δ+ http://en.wikipedia.org/wiki/Alpha_helix

6. If a little is good, more is better

7. Lower cysteine pKa is not always correlated with enhanced reactivity At pH=7.4: 1. • A cys with pKa=6.5 is 89% ionized • A cys with pKa=5.5 is 98.8% ionized • A cys with pKa=2.0 is 99.9996% ionized Henderson-Hasselbalch predicts exponentially diminishing returns as pKa is depressed below physiological pH: more is not (much) better 2. The Bronsted Catalysis Law dictates that lower pKa cysteines are less reactive: more is worse

8. The Counterintuitive Result of Bronsted Catalysis Law Rate of DTNB reduction by various thiols has an optimum when pKa is close to pH Bronsted catalysis law: From transition state theory: Whitesides et al., J. Org. Chem, 1977 Conjugate bases of high pKa acids are “harder” nucleophiles and more reactive

9. How to measure cysteine pKa in proteins

10. Thiolates have enhanced UV absorption Thiolates absorb ~240 nm light due to n->σ* lone pair transitions Strengths: direct detection, simple equipment, quantitative Weaknesses: requires control experiment to ensure that cysteine of interest is being monitored, tyrosine ionization Noda et al., JACS, 1953 Note: 3 is n-butylmercaptan in 1 N NaOH, 4 is same in 0.01 N HCL

11. Thiolates react rapidly with electrophiles Rate of cysteine modification as a function of pH Strengths: potentially large signal, multiple means of detection Weaknesses: Steric effects can be problematic, extreme pH values can effect probes In this study, steric effects were problematics Nelson et al., Biochemistry, 2008

12. Thiolates result in perturbed chemical shifts for nearby nuclei in NMR Strengths: direct detection, information about other ionizations Weaknesses: requires pure isotopically labeled sample, size limit (mass<35 kDa), confounding chemical shift effects

13. Application to DJ-1 • Absence causes Parkinson’s disease • Overexpression associated with multiple cancers • Absence exacerbates repurfusion injury (stroke) • Protects against mitochondrial damage and resulting fission The protective function of DJ-1 requires a conserved cysteine residue (C106)

14. C106 is in a solvent exposed pocket Witt et al., Biochemistry, 2008

15. A protonated glutamic acid depresses C106 pKa in DJ-1 E18 C106 1.2 Å resolution, 5.0σ 2FO-FC Witt et al., Biochemistry, 2008

16. Substitutions at E18 is raise C106 pKa Note: Even in E18L, C106 is still a low pKa cysteine Witt et al., Biochemistry, 2008

17. Proximal arginines bind an anion and raise C106 pKa Witt et al., Biochemistry, 2008

18. Summary • Cysteine thiolates are stabilized by positive charges, the helix macrodipole, and hydrogen bonding • The most reactive cysteines have pKa values near physiological pH • UV spectroscopic, NMR and rapid kinetic approaches can be used to determine cysteine pKa values • Caution must be used in assessment of structural determinants of cysteine reactivity-incompletely understood