Q22M, T44W, R81G, H83G, T84M, N130G, N172M, A234S, T236L, C9G, V48G, L50W, T78S, S101E, E237M, T265S, W267F 7, 11, , 239,
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1 111 Table 4-1. Summary of design calculations for Kemp elimination enzymes. The residues allowed for each required catalytic contact are indicated along with the actual catalytic residue chosen from the active site search in parentheses. The positions allowed to change identity during the active site search and repacking calculations are indicated as design residues. Float residues are allowed to change conformation but not identity. required contacts, allowed residues (actual catalytic contact) repacking residues design scaffold base!-stacking H-bond active site search design residues design float mutations HG-2 1GOR D (D126) F/W (T44W) S/T/Y (S265T) 17, 42, 44, 81, 83, 126, 129, 170, 172, 209, 234, 236, 237, 265, , 21, 81, 83, 84, 125, 130, 172, 234, 236, , 17, 46, 47, 50, 79, 87, 90, 170, 207, 209, 239, Q22M, T44W, R81G, H83G, T84M, N130G, N172M, A234S, T236L, E237M, T265S, W267F 1THF-1 1THF E (S101E) F/W (L50W) K (L222K) 7, 9, 11, 48, 50, 101, 126, 128, 130, 169, 171, 176, 199, 201, 222, 224 9, 48, 78, 101, 103, 126, 128, 130, 169, 171, 199, 201 7, 11, 176 C9G, V48G, L50W, T78S, S101E, N103M, Q128W, D130A, L169A, T171V, I199G, S201M, L222K 1THF-2 1THF E (S101E) F/W (L50W/A128W) S/T/Y (S201) " 9, 48, 78, 126, 128, 130, 169, 171, 222 7, 11, 176, 199, 224 C9G, V48G, L50W, T78S, S101E, A128W, D130A, L169A, T171L, L222V 1A53-1 1A53 E (L231E) F/W (K110W) S/T/Y (L131S) 51, 81, 83, 89, 108, 110, 112, 131, 133, 157, 159, 178, 180, 182, 184, 210, 211, , 81, 108, 157, 159, 180, , 89, 112, 133, 178, 182, 184, 211 E51I, S81G, K110W, L131S, L157G, E159W, N180G, E210S, L231E 1A53-2 1A53 E (G178E) F/W (K110W/E210W) none " 51, 81, 83, 108, 131, 157, 159, 180, 211, 231 8, 53, 89, 108, 112, 133, 182, 184 E51A, S81A, L83A, K110W, L131A, K157A, E159V, G178E, N180A, E210E, S211Q, L231G 1A53-3 1A53 E (L157E) F/W (K110W/E210W) none " 81, 83, 108, 131, 178, 180, 211, 231 8, 53, 89, 112, 129, 133, 182, 184 L51E, S81A, L83A, K110W, L131A, L157E, E159I, N180M, E210W, S211Q, L231G
2 Table 4-2. Physical characteristics of protein variants. 112 molecular weight (g/mol)! (M -1 cm -1 ) formal charge # amino acids 1THF KE THF THF A KE A A A GOR HG
3 Table 4-3. Experimental characterization of designed Kemp elimination enzymes. 113 parent mutation scaffold k cat (s -1 ) K m (mm) k cat /K m (s -1 M -1 ) a k cat /k uncat T m ( C) HG-2-1GOR NA NA 130 NA 46.6 HG-2 D127N 1GOR NA NA 2 NA 51.0 HG-2 S265A 1GOR NA NA 54 NA 45.8 HG-2 K50A 1GOR NA NA 37 NA 49.3 HG-2 G81A 1GOR NA NA 17 NA 46.7 HG-2 S265T 1GOR 0.68 ± ± E A A ± ± E+04 ND 1A A ± ± E+04 ND 1THF-2-1THF NA NA 5 NA 73.7 KE07 b, c - 1THF ± ± E+04 NA KE07 b, d - 1THF ± ± E+04 ND KE70 b, c - 1JCL 0.16 ± ± E+05 NA KE70 b, d - 1JCL ± ± E+04 ND ND = not determined NA = not applicable a k uncat under the assay conditions was determined to be 1.16x10-6 s -1 by Rothlisberger et al. b These enzymes were designed by Rothlisberger et al. c The kinetic constants were determined by Rothlisberger et al. d The kinetic constants were determined as a part of this thesis work.
4 Table 4-4. Design summary of Kemp elimination enzymes. MD prediction and experimental detection of activity are indicated. 114 # mutations activity name scaffold base!-stack H-bond from wt from active design MD experimental KE59 a 1A53 L231E K110W L131S 10 1A53-1 1A53 L231E K110W/E159W L131S 9 8 (KE59) no no KE10 a 1A53 G178E E210W A53-2 1A53 G178E K110W/E210W (KE10) yes yes 1A53-3 1A53 L157E K110W/E210W yes yes KE07 a 1THF S101E L50W L222K 13 1THF-1 1THF S101E K50W/A128W L222K 13 9 (KE07) yes no 1THF-2 1THF S101E L50W/A128W S (KE07) yes yes HG-2 1GOR D126 T44W T265S 12 - yes yes HG2-S265T 1GOR D126 T44W T (2.2.0) increase higher k cat /K m HG2-G81A 1GOR D126 T44W T265S 13 1 (2.2.0) decrease decrease HG2-K50A 1GOR D126 T44W T265S 13 1 (2.2.0) decrease decrease a These KE enzymes were designed by Rothlisberger et al.
5 115 A Figure 4-1. Active site locations of first- and second-generation designs in TAX. (A) The active site of first generation design (purple) is in the native binding pocket for xylobiose (grey). () The active site of second-generation design HG-2 (yellow) is located deeper into the barrel of the scaffold. A Figure 4-2. Active site of HG-2. (A) Catalytic residues identified through the active site search. () Repacked active site (pink) overlaid with the wild-type residues (grey).
6 Figure 4-3. Active site structures of HG-2 during the MD simulation. Three active site configurations are shown: the initial configuration, a representative active site from state 1, and a representative active site from state
7 117 A Figure 4-4. Distance distributions of HG-2. (A) Catalytic site RMSD with respect to the initial configuration as the simulation progresses. () Distance distributions for important catalytic contacts.
8 118 A C C Figure 4-5. Expression and purification of HG-2. (A) SDS-PAGE gel of HG-2 expression and purification. Lane 1: MW marker. Lanes 2 and 3: uninduced cells. Lanes 4 and 5: cells 18 hours after IPTG induction. Lane 6: lysate supernatant. Lane 7: lysate pellet. Lane 8: Ni-NTA purification elution. () Electrospray mass spec. Expected mass: , actual mass: (C) UV image of HG-2 crystals grown in 0.1 M MES, ph 6.5, 1.6 M Magnesium Sulfate with 9.5 mg/ml HG-2.
9 119 A wt TAX HG-2 wt TAX HG-2 Figure 4-6. CD analysis of wild-type TAX and HG-2. (A) Far-UV wavelength scan. () Thermal denaturation. All experiments were carried out with 10 µm protein in 25 mm HEPES ph 7.25, 100 mm NaCl.
10 120 A background wt TAX HG-2 KE70 HG2 HG2-S265A HG2-D127N Figure 4-7. Kinetic characterization of second-generation enzymes. 5 µm protein is used in 25 mm HEPES ph 7.25, 100 mm NaCl, 27 C. (A) Michaelis-Menten plots of design HG-2 (red), wild-type TAX (blue) and background reaction (black). () Michaelis-Menten plots of KE70 (red), HG-2 (blue), and HG-2 knock out mutants HG-2- S265A (green) and HG-2-D127N (yellow).
11 A 121 Figure 4-8. Effect of ph on the activity and structure of HG-2. (A) ph-rate profile of HG-2. () Far-UV CD wavelength scan of HG-2 at various phs.
12 122 KE70 HG2 HG2-S265T HG2-G81A HG2-K50A Figure 4-9. Kinetic characterization of third-generation enzymes. Michaelis-Menten plots of KE70 (red), design HG-2 (blue), 220-S265T (green), G81A (yellow), and 220- K50A (grey). 5 µm protein was used and the reactions were carried out in 25 mm HEPES ph 7.25, 100 mm NaCl, 27 C.
13 123 A C D E F Figure Active sites of designs in scaffold 1A53. (A) Catalytic residues of 1A53-1 (pink) overlaid with those from KE59 (grey). () Repacked active site of 1A53-1. (C) Catalytic residues of 1A53-2. (D) Repacked active site of 1A53-2. (E) Catalytic residues of 1A53-3. (F) Repacked active site of 1A53-3.
14 124 KE07 1A53-2 1A53-3 1THF-2 Figure Kinetic characterization of designs in scaffolds 1A53 and 1THF. Standard errors are calculated from three measurements.
15 125 A C D Figure Active sites of designs in scaffold 1THF. (A) Catalytic residues of 1THF1 (blue) overlaid with those from KE07 (grey). () Repacked active site 1THF-1. (C) Catalytic residues of 1THF-2 (yellow) overlaid with those from KE07 (grey). (D) Repacked active site of 1THF-2.
16 126 A C Figure MD analysis of 1A53-1. (A) Initial active site configuration of 1A53-1. () Representative configuration after MD simulation. (C) Distance distributions of contacts between the substrate and catalytic residues.
17 127 A C D E F Figure MD analysis of 1A53-2. (A) Initial active site configuration of 1THF-1. () Representative configuration midway through MD simulation (12 ns). (C) Representative geometry after MD simulation. (D) Number of water molecules that enter the active site over the course of the simulation. (E) Distance distribution of substrate-base contact. (F) Distance of the substrate-base contact over the course of the simulation.
18 128 A C Figure MD analysis of 1A53-3 (A) Initial active site configuration of 1A53-3. () Representative configuration after MD simulation. (C) Distance distributions of contacts between the substrate and catalytic residues.
19 129 A C Figure MD analysis of 1THF-1. (A) Initial active site configuration of 1THF-1. () Representative configuration after MD simulation. (C) Distance distributions of contacts between the substrate and catalytic residues.
20 130 A C Figure MD analysis of 1THF-2. (A) Initial active site configuration of 1THF-2. () Representative configuration after MD simulation. (C) Distance distributions of contacts between the substrate and catalytic residues.
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