Principles of Cysteine Reactivity in Proteins

Mark Wilson Redox Biology Center

Department of Biochemistry University of Nebraska-Lincoln

Redox Course June 10th, 2019

What is the origin cysteine reactivity?

Cysteine thiolates are the reactive species

pKa~8-9

Thiolates are potent nucleophiles that will react with most electrophiles

Thermodynamics of cysteine ionization

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Ka =H +[ ] S−[ ]HS[ ]

pKa = −log10Ka

lnKa = −ΔG0

RT= −

ΔH 0

RT+ΔS 0

R

Ka is the acid dissociation equilibrium constant

The van’t Hoff relationship allows a direct link between Keq and thermodynamics (ΔG0)

Relating the pH to cysteine ionization

pKa = − log10H +⎡⎣

⎤⎦ S

−⎡⎣

⎤⎦

SH[ ]

⎛

⎝

⎜⎜

⎞

⎠

⎟⎟

pKa = − log10 H+⎡⎣ ⎤⎦− log10

S−⎡⎣ ⎤⎦SH[ ]

pKa = pH − log10S−⎡⎣ ⎤⎦SH[ ]

pH − pKa = log10S−⎡⎣ ⎤⎦HS[ ]

Henderson-Hasselbalch equation

At pH=7.5, a pKa=8.5 thiol is about 10% ionized

Cysteine pKa depression is a matter of diminishing returns

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pH − pKa = log10S−[ ]HS[ ]

•  A cys with pKa=6.0 is 90% ionized •  A cys with pKa=5.0 is 99% ionized

•  A cys with pKa=2.0 is 99.999% ionized

At pH=7.0:

L.B. Poole, Free Radic Biol Med. 2015

Red slider shows proportion of thiolate for a

Cys with pKa=6.0

The kinetics of cysteine reactions are directly proportional to pKa value

Whitesides et al., J. Org. Chem, 1977

Rate of DTNB reduction by various thiols has an optimum when pKa is close to pH

Brønsted relation:

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log10 k = β ∗ pKa + C

k∝ e−ΔG±

RT ; K = e−ΔGRT

From transition state theory:

Conjugate bases of high pKa acids are “harder” nucleophiles and more reactive

Cys with pKa~pH are most reactive a priori

Methods of measuring Cys pKa values

Witt et al., Biochemistry 47 (28) 2008

•  Thiolates absorb 240 nm light more than thiols

•  Other methods for Cys

pKa determination include pH-dependent reaction rates and NMR

Filled circles: WT DJ-1, open squares: C106S DJ-1

How do proteins stabilize cysteine thiolates?

Structural determinants of cysteine pKa depression: electrostatics

Cationic residues (Lys, Arg) have flexible sidechains-fluctuating fields

Histidine imidazolium is best suited for stable electrostatic

interactions with thiolate

How can we distinguish between electrostatic and

H-bonding effects?

catalytic dyad

Structural determinants of cysteine pKa depression: Hydrogen bonding

Charge-assisted hydrogen bonds (CAHB) are always more stable: they pull thiols towards thiolates

δ+

δ-

ΔG1>ΔG2

1 2

Hydrogen bonding is the dominant contributor to cysteine pKa depression

Roos et al., Antioxidants and Redox Signaling (2013)

Cys32 pKa=6.7 Cys30 pKa=3.5 Cys11 pKa~4-5

The α-helix macrodipolar effect is likely just H-bonding δ+ δ-(?)

Roos et al., Antioxidants and Redox Signaling (2013)

H-bond polarization can significantly decrease Cys pKa values

Madzelan et al., FEBS Journal (2012)

T114V mutation increases C111 pKa from 4.6 to 5.2

Better H-bond donor

A protonated glutamic acid depresses Cys106 pKa in DJ-1

Witt et al., Biochemistry 47 (28) 2008

C106

E18

1.2 Å resolution, 5.0σ 2FO-FC

Protonated acidic residues lower cysteine pKa values in diverse enzymes

Lim et al., JBC, 287 (30), 2012

C72-E115 of Methionine sulfoxide reductase A

C1-D422 of Mtu RecA intein Du et al., JACS, 133 (26), 2011

Good H-bond donor

Beware of “obvious” structural arguments for Cys pKa depression

Witt et al., Biochemistry 47 (28), 2008

1.2 Å resolution Rwork=14% Rfree=18%

pKa=5.4

pKa=5.2

pKa=5.0

What happens to reactive cysteine residues in the cell?

An (incomplete?) inventory of cysteine modifications

Chung et al, (2015), Circulation Research

H2O2 is a popular substrate

100-200 nM basal concentration in the cell Peroxiredoxins (50-500x more abundant than the other enzymes): kcat/KM ~107 M-1 s-1 Catalases: kcat/KM~108 M-1 s-1 Glutathione peroxidases (Gpx): kcat/KM ~107 M-1 s-1

If you want to react with H2O2, you must be fast

Kinetic versus thermodynamic reasoning in cysteine biochemistry

Thermodynamics: What can happen (i.e. is possible)? Gibbs free energy (ΔG), equilibrium constant (Keq) Kinetics: What will happen on biologically relevant timescales? kobs, kcat

A given cysteine residue can react with many species, but but kinetic competition determines its fate in the cell

Which modification will be observed on a given cysteine residue?

We can’t yet predict, but enzyme-based reasoning is needed for a kinetic perspective

P.A. Karplus, (2014) Free Radical Biology and Medicine

Transition state stabilization by Prx: A general model for Cys

sulfenylation?

A kinetic perspective on GAPDH Cys oxidation

GAPDH is a prominent target of H2O2, re-directing carbon flux to the pentose phosphate pathway to make NADPH

Peralta et al, Nat. Chem. Biol., (2015).

Summary •  Thiolates are the reactive ionization state of cysteine •  Hydrogen bonding in the dominant mechanism of depressing Cys pKa values •  Cys pKa is a poor a priori predictor of reactivity

•  Cysteine in proteins is subject to diverse modifications, directed by the structural microenvironment •  Kinetic, rather than thermodynamic, considerations should guide our thinking about cysteine fate in the cell