The Physical Biochemistry of Thiol Ionization Mark Wilson May 21 st , 2009
The Physical Biochemistry of Thiol Ionization
Mark Wilson
May 21st, 2009
Cysteine pKa values must be depressed for thiolate formation
pKa~8-9
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Ka =H +[ ] S
−[ ]
HS[ ]pKa = −log10Ka
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pH − pKa = log10S−[ ]
HS[ ]
Henderson-Hasselbalch equation
At pH=7.4, about 7% of thiol is ionized€
lnKa = −ΔG
RT
Electrostatic thiolate stabilization
Positive charges (e.g. lysine, arginine, metals) will electrostatically stabilize thiolate anion formation
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U =q1q24πεoκr
Coulomb potential energy
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
r is distance, q is charge; always pairwise additive
Hydrogen bonding to the thiolate
Hydrogen bond donation to cysteine commonly lowers pKa
The α-helix macrodipole
http://en.wikipedia.org/wiki/Alpha_helix
C; δ-
N; 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
The peptide dipole
Only the first turn contributes significantly to pKa depression
If a little is good, more is better
Lower cysteine pKa is not always correlated with enhanced reactivity
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pH − pKa = log10S−[ ]
HS[ ]
• 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
At pH=7.4:
Henderson-Hasselbalch predicts exponentially diminishing returns as pKa is depressed below physiological pH: more is not (much) better
The Bronsted Catalysis Law dictates that lower pKa cysteines are less reactive: more is worse
1.
2.
The Counterintuitive Result of Bronsted Catalysis Law
Whitesides et al., J. Org. Chem, 1977
Rate of DTNB reduction by various thiols has an optimum when pKa is close to pH
Bronsted catalysis law:
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log10 k = β ∗pKa +C
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k ∝ e−ΔG ±
RT ;K = e−ΔG
RT
From transition state theory:
Conjugate bases of high pKa acids are “harder” nucleophiles and more reactive
How to measure cysteine pKa in proteins
Thiolates have enhanced UV absorption
Noda et al., JACS, 1953
Note: 3 is n-butylmercaptan in 1 N NaOH, 4 is same in 0.01 N HCL
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
Thiolates react rapidly with electrophiles
Rate of cysteine modification as a function of pH
Nelson et al., Biochemistry, 2008
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
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
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)
C106 is in a solvent exposed pocket
Witt et al., Biochemistry, 2008
A protonated glutamic acid depresses C106 pKa in DJ-1
Witt et al., Biochemistry, 2008
C106
E18
1.2 Å resolution, 5.0σ 2FO-FC
Substitutions at E18 is raise C106 pKa
Witt et al., Biochemistry, 2008
Note: Even in E18L, C106 is still a low pKa cysteine
Proximal arginines bind an anion and raise C106 pKa
Witt et al., Biochemistry, 2008
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