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 isomerase Jason P. Schwans, Daniel A. Kraut, and Daniel Herschlag PNAS, vol. 106, (2009) Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole Daniel A. Kraut, Paul A. Sigala, Timothy D. Fenn, and Daniel Herschlag PNAS, vol. 107, (2009)

Ketosteroid isomerase (KSI) Bacterial Δ 5 -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 Tyr16 Remote binding

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase Jason P. Schwans, Daniel A. Kraut, and Daniel Herschlag PNAS, vol. 106, (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 rings Full-length (S full ): 5(10)-estrene-3,17-dione Miniature (S mini ): 3-cyclohexen-1-one PDB: 1OH0 PDB: 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 S full S mini k HO- M -1 s ±0.01×10 -1 k AcO- M -1 s ±0.1× ±1×10 -7 Reactivity of substrates are nearly identical.

Overall contribution of distal rings S full S mini Ratio S full /S mini k cat /K M M -1 s ±0.7× ±0.2× ,000 k cat s ±0.25.5± Rate constant of WT KSI

Hole mutations S full S mini Ratio S full /S mini D103N2.0±0.4× ±0.9× ,000 D103L 1.8±0.2× ±0.1× ,000 Y16S 1.9±0.7× ±0.1× ,000 Y16F 8.0±1.0× ±0.3× ,000 k cat /K M M -1 s -1

Hole mutations S full S mini Ratio S full /S mini D103N2.9±0.1× ±0.3× D103L 3.9±0.3× ±0.2× Y16S 3.9±0.2× ±1.0× Y16F 5.4±0.2× ±0.3× K cat 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 10 4 fold predicted difference in catalysis, which is contrary to the similar catalysis (k cat ) of bound single- and multi-ringed substrate

Conclusions 1.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 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: K cat and K M 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 ablated 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 analogue  Not much structural change identified

Fig. 3(A) Structural studies of KSI. Sigma-A-weighted 2F o − F c 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 analogue  No other H-bonding between equilenin and other residues Cavity

Model:Tyr16Ser mutation replaces Tyr16 with a water-occupied cavity 1.100Å 3 cativity 2.Polar contacts in the cavity 3.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 Significant chemical shift differences of the fluorine nucleus in Tyr16Phe versus Tyr16Ser 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