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