Table 1.
Enzymatic activity and nature of product of mutant T4L
| Mutant | Active site residues*
|
Activity, halo assay | Anomeric form of product | ||
|---|---|---|---|---|---|
| 11 | 20 | 26 | |||
| WT* | Glu | Asp | Thr | +++ | α |
| T26D | Asp | + | n.d. | ||
| T26E | Glu | − | Covalent adduct† | ||
| T26Q | Gln | − | n.p. | ||
| T26H | His | ++ | β | ||
| D20C | Cys | Thr | +++ | α | |
| D20C/T26D | Cys | Asp | + | n.d. | |
| D20C/T26E | Cys | Glu | − | Covalent adduct† | |
| D20C/T26H | Cys | His | ++ | β | |
| D20S | Ser | Thr | + | n.d. | |
| D20T | Thr | + | n.d. | ||
| D20A | Ala | ++ | α | ||
| D20E | Glu | ++ | α | ||
| D20N | Asn | − | n.p. | ||
| D20N/T26D | Asn | Asp | − | n.p. | |
| D20N/T26E | Asn | Glu | − | n.p. | |
| E11D | Asp | Asp | Thr | ++ | α |
| E11D/T26E | Asp | Glu | − | Covalent adduct† | |
| E11Q | Gln | Asp | Thr | − | n.p. |
| E11Q/T26E | Gln | Glu | − | n.p. | |
| E11N | Asn | Asp | Thr | − | n.p. |
| E11N/T26E | Asn | Glu | − | n.p. | |
| E11H | His | Asp | Thr | − | n.p. |
| E11H/T26E | His | Glu | − | n.p. | |
| E11H/T26H | His | His | − | n.p. | |
| E11H/D20C | His | Cys | Thr | − | n.p. |
n.d.: Could not be detmined; n.p.: no product.
*
Unnamed residues are as in WT.
†
Mutants T26E, D20C/T26E, and E11D/T26E give double bands when purified by SDS/PAGE. In the case of T26E this was shown by MS to be caused by a mixture of the mutant protein plus the covalent adduct. The presence of the adduct is inferred but was not verified directly for D20C/T26E or E11D/T26E.