Catalytic role of remote substrate binding and active-site hydrogen bonding in ketosteroid

isomerase

Josh Demeo, Yin Wong, Cuiwen He, and Yang Liu

April 26, 2012

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomeraseJason P. Schwans, Daniel A. Kraut, and Daniel Herschlag

PNAS, vol. 106, 14271-14275 (2009)

Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion holeDaniel A. Kraut, Paul A. Sigala, Timothy D. Fenn, and Daniel Herschlag

PNAS, vol. 107, 1960-1965 (2009)

Ketosteroid isomerase (KSI)

Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate

Biochemistry, 1997, 36 (46), pp 14030–14036

PDB: 1OH0

KSI substrate and its enzymatic reaction

Substrate (Full length):5(10)-estrene-3,17-dione

KSI

Monitor the reaction with ΔA246 Initial rates at different [S] Michaelis-Menten Kinetics

Reaction mechanism

Tyr16

Asp103

Asp40

Tyr16Remote binding

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomeraseJason P. Schwans, Daniel A. Kraut, and Daniel Herschlag

PNAS, vol. 106, 14271-14275 (2009)

Different from chemical catalysts, enzymes can use binding interactions with nonreactive portions of substrates to accelerate chemical reactions.

Reaction Mechanism

Substrates w/ or wo/ distal ringsFull-length (Sfull):

5(10)-estrene-3,17-dioneMiniature (Smini):

3-cyclohexen-1-one

PDB: 1OH0PDB: 1PZV

Potential impacts of distal rings on enzymatic catalysis

• Differences in substrates reactivity

• Remote binding between distal rings and KSI

• Transition state stabilization

• Positioning the steroid relative to the oxyanion hole

• Removal of the oxyanion hole water molecules

• Influence the electrostatic behavior of oxyanion hole

Non-enzymatic isomerization

Sfull Smini

kHO-

M-1s-1 1.7±0.01×10-1 1.7±0.01×10-1

kAcO-

M-1s-1 1.1±0.1×10-6 6.5±1×10-7

Reactivity of substrates are nearly identical.

Overall contribution of distal rings

Sfull SminiRatio

Sfull/Smini

kcat/KM

M-1s-1 5.4±0.7×105 2.0±0.2×101 27,000

kcat

s-1 9.0±0.2 5.5±0.5 1.6

Rate constant of WT KSI

Hole mutations

Sfull SminiRatio

Sfull/Smini

D103N 2.0±0.4×104 4.5±0.9×10-1 44,000

D103L 1.8±0.2×103 1.2±0.1×10-115,000

Y16S 1.9±0.7×103 9.2±0.1×10-221,000

Y16F 8.0±1.0×100 6.8±0.3×10-412,000

kcat/KM M-1s-1

Hole mutations

Sfull SminiRatio

Sfull/Smini

D103N 2.9±0.1×10-1 2.0±0.3×10-1 1.5

D103L 3.9±0.3×10-2 1.5±0.2×10-22.6

Y16S 3.9±0.2×10-2 2.3±1.0×10-21.7

Y16F 5.4±0.2×10-4 3.4±0.3×10-41.6

Kcat s-1

Electrostatic complementarity…•Electrostatic complementarity for the oxyanion hole provides only a

modest contribution according to Kraut et. al

• Currently want to look at single-ring phenolate transition state analogs

to see if they accurately reflect the change in oxyanion hole

electrostatics of multi-ring phenolates

•Recent computational results by Warshal et. al:

• Showed greater electrostatic contribution to reaction of a full length

steroid (2 kcal/mol) versus a single-ring substate (8 kcal/mol)

• This corresponds to a 104 fold predicted difference in catalysis,

which is contrary to the similar catalysis (kcat) of bound single- and

multi-ringed substrate

Conclusions1. Binding interactions with the distal steroid rings provides ~5 kcal/mol to catalysis

2. Catalysis is the rate-limiting step

3. Some catalysis is attributed to the oxyanion hole hydrogen bonds

4. Positioning and/or ground state destablization of the active site Asp general base

may provide an additional rate advantage

5. Local interactions position the substrate within the oxyanion hole and binding

interactions and solvent exclusion by the distal steroid rings contribute little to

determining oxyanion hole energetics

6. Remote steroid rings help localize the substrate to the active site and interactions

with proximal rings position the substrate with respect to the active site general

base and oxyanion hole hydrogen bonds

7. There may be some modest contribution by these hydrogen-bonds towards the

geometrical optimization of the transition state interactions

Paper 2.

Question addressed

• Reduction of catalysis by Y16F (6.3kcal/mol) >> effects of H-bond mutations in other enzymes

• Explanation: H-bond formation with substrate > with water 6kcal/mol

However,• Conservative Y16F impairs catalysis much more than less-

conservative Y16S (reported to be 50000 folds and 30 folds separately)

• Paradoxical effects of mutations yet to be answered

Summary

• Moderate rate reduction in Y16S is not a result of maintenance of H-bond in residue-16.

• H-bond cannot be formed between Ser16 with intermediate analogue.

• Y16F does not allow water to fill into the oxyanion hole yet Y16S does.

• The aqueous-like solvation of intermediate in Y16S may account for moderate rate reduction.

• Previous study proposed Ser-16 maintained H-bonding ability of residue 16– Expect residue-16 mutants without hydroxyl group with larger decrease

Method:Kcat and KM of pKSI WT and mutant using substrate 5(10)-EST

Results:Moderate rate reduction seen also in pKSI mutants without hydroxyl group for hydrogen bonding

Maintenance of H-bonding ability does not explain Moderate reduction

abla

ted

abla

ted

But does mutation causes stabilization by any other means?

Fig. 3(B) Superposition of the pKSI Tyr16Ser∕Asp40Asn · equilenin structure determined herein (carbon atoms colored cyan), the 1.8-Å resolution structure of unliganded pKSI Tyr16Phe (PDB entry 1EA2, carbon atoms colored violet), and the 1.1-Å resolution structure of equilenin bound to wild-type pKSI (PDB entry 1OH0, carbon atoms colored green).

Results:Orientation of bound equilenin to pKSI Y16S mutant nearly identical to that of wild type

H-bond between residue 16 with equilenin ablated by mutation Y16S

H-bonding is not present between Ser16 and analogueNot much structural change identified

Fig. 3(A) Structural studies of KSI. Sigma-A-weighted 2Fo − Fc electron density map (contoured at 1.5σ) from the 1.6-Å resolution structure of equilenin bound to Tyr16Ser/Asp40Asn pKSI. Distances are average values (standard deviation 0.1 Å) from the four independently refined monomers contained in the asymmetric unit.

X-ray structure of equilenin , intermediate analogue, binding to Y16S mutant

Results:Ser16 sidechain H-bonded with Met13 instead

O---O distance (16Ser hydrozyl-equilenin oxygens) :6.4Å >> 3.5Å H-bonding distance+ no other enzymatic group positioned

H-bonding is not present between Ser16 and analogueNo other H-bonding between equilenin and other residues

Cavity

Model:Tyr16Ser mutation replaces Tyr16 with a water-occupied cavity

1. 100Å3 cativity2. Polar contacts in the

cavity3. Triangle-shaped

electron density in the 2Fo-Fc electron density map.

4. Greatest electron density is located 3.0 Å and 2.7 Å from the oxygens of equilenin and Tyr57.

5. Nearly identical rate effects of the Tyr16 to Ser, Thr, Ala, and Gly mutations.

Consequences of the Tyr16Phe mutation

local solvation environment within the oxyanion hole near residue 16

1. Significant chemical shift differences of the fluorine nucleus in Tyr16Phe versus Tyr16Ser.

2. 2-fluoro-4- nitrophenolate in water and in the aprotic, low polarity solvent tetrahydrofuran (THF) .

Conclusions

Further studies

• Philip Hanoian and Sharon Hammes-Schiffer. Water in the active site of ketosetroid isomerase. Biochemistry, 2011, 50 (31), pp 6689–6700.

• Jason P. Schwans, Fanny Sunden, Ana Gonzalez, Yingssu Tsai, and Daniel Herschlag. Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase. J. Am. Chem. Soc., 2011, 133 (50), pp 20052–20055