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

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

Electrostatic thiolate stabilization Positive charges (e.g. lysine, arginine, metals) will electrostatically stabilize thiolate anion formation 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 pK a

The α-helix macrodipole C; δ - N; δ + 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 pK a depression

If a little is good, more is better

Lower cysteine pK a is not always correlated with enhanced reactivity A cys with pK a =6.5 is 89% ionized A cys with pK a =5.5 is 98.8% ionized A cys with pK a =2.0 is % ionized At pH=7.4: Henderson-Hasselbalch predicts exponentially diminishing returns as pK a is depressed below physiological pH: more is not (much) better The Bronsted Catalysis Law dictates that lower pK a 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 pK a is close to pH Bronsted catalysis law: From transition state theory: Conjugate bases of high pK a acids are “harder” nucleophiles and more reactive

How to measure cysteine pK a 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 pK a in DJ-1 Witt et al., Biochemistry, 2008 C106 E Å resolution, 5.0σ 2F O -F C

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

Proximal arginines bind an anion and raise C106 pK a Witt et al., Biochemistry, 2008

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