Wild type | Significance | Mutant | Enzyme activity2 | Properties of mutant proteins | |||
---|---|---|---|---|---|---|---|
residues | Â | proteins | (%) | Km | Vmax | Kcat/Km | Â |
Control | Â | Â | 100 | 59.5 | 110 | 74 | Â |
D263 | Important residue for substrate binding; forms H-bonds with All OH groups of tricetin | D263E | 4.01 | Â | Â | Â | Severe loss of activity is due to a conflict between the catalytic His262-imidazole group and Glu-CH2 |
 |  | D263I | 0.08 |  |  |  | Ile263 can not form a H-bond with 3'-OH group |
 |  | D263N | 84.33 | 128.2 | 20.3 | 63 | Slight decrease in activity due to a decreased electronegativity of Asn-N compared to Asp-O, that affects charge transfer to tricetin-OH groups |
E290 | H-bonds with tricetin 4'-OH; forms an H-bonding network with neighboring residues, esp. E290-COO- and H262-backbone-NH | E290I | 1.7 | Â | Â | Â | Loss of activity is due to the fact that Ile can not form a H-bond with the 4'-OH of tricetin |
 |  | E290Q | 0.06 |  |  |  | This mutation results in a more extensive H-bonding that hinders charge transfer and affects B-ring flexibility |
W259 | H-bonds with selgin 4'-OH and forms a H-bonding network with neighboring residues | W259A | 79.23 | 131.0 | 17.02 | 5.20 | Ala can maintain the H-bonding network between Trp259, Glu290 and His262, wheras Tyr cannot |
 |  | W259Y | 49.13 | 170.1 | 9.90 | 2.33 |  |
E322 | H-bonds with tricetin 5'-OH. | E322I | 54.33 | 193.17 | 30.56 | 6.33 | Loss of charge or a change in the side chain affects H-bonding with the neighboring residues, especially His262 |
 |  | E322Q | 40.3 |  |  |  |  |
G305 | H-bonds with selgin 7-OH; important residue for substrate positioning | G305S | 63.33 | 118.41 | 21.65 | 7.31 | Change in polarity is less effective than chain length on catalytic activity. |
 |  | G305A | 0.14 |  |  |  | Loss of activity due to loss of H-bonding with the amide group of the neighboring Asn348 |
N124 | H-bonds with O-4/O-5 of all substrates in order to orient them to the most favorable position | N124I | 1.8 | Â | Â | Â | Resuled in a decreased substrate binding but not protein folding. Both mutations disrupt H-bonding with 5-OH group of tricetin |
 |  | N124Q | 4.1 |  |  |  |  |
H262 | Putative catalytic base involved in deprotonation of tricetin hydroxyl groups | H262R | 0.01 | Â | Â | Â | Resulted in almost complete loss of protein expression; all mutant proteins lack imidazole ring that is critical for proton flow among His262, Asp263 and the substrate |
 |  | H262L | 0.96 |  |  |  |  |
 |  | H262F | 1.06 |  |  |  |  |