Table 1. Kinetic parameters of IQF peptide cleavage by rOaVPE1b.
| IQF peptide | Sequence* | Vmax (nmoles min-1 mg-1 protein) (±s.e.m.)† | Km (μM) (±s.e.m.)† | kcat (min-1) (±s.e.m.)†,‡ |
|---|---|---|---|---|
| wt | Abz-STRN↓GLPS-Y(3NO2) | 51.3 (±5.8) | 55.0 (±6.4) | 1.6 (±0.2) |
| R28A | Abz-STAN↓GLPS-Y(3NO2) | 6.9 (±0.6) | 13.0 (±2.4) | 0.2 (±0.02) |
| R28K | Abz-STKN↓GLPS-Y(3NO2) | 29.3 (±3.5) | 42.0 (±4.0) | 0.9 (±0.1) |
| N29A | Abz-STRA↓GLPS-Y(3NO2) | NA§ | − | − |
| N29Q | Abz-STRQ↓GLPS-Y(3NO2) | NA§ | − | − |
| N29D | Abz-STRD↓GLPS-Y(3NO2) | ∼2|| | ND|| | ∼0.06|| |
| G30A | Abz-STRN↓ALPS-Y(3NO2) | 51.0 (±2.0) | 29.0 (±1.4) | 1.6 (±0.07) |
| G30S | Abz-STRN↓SLPS-Y(3NO2) | 35.5 (±2.7) | 31.4 (±2.5) | 1.1 (±0.08) |
| L31A | Abz-STRN↓GAPS-Y(3NO2) | NA§ | − | − |
| L31I | Abz-STRN↓GIPS-Y(3NO2) | ND¶ | ND¶ | ND¶ |
IQF, internally quenched fluorescent; NA, no activity; ND, not determined.
*IQF peptide residues are numbered according to their position within the native kB1 precursor, where the mature cyclotide incorporates Gly1-Asn29; native Cys26 is substituted with Ser to avoid unpaired Cys residues.
†N≥ 3;±standard error of the mean (s.e.m.)
‡kcat is a conservative estimate assuming that the total concentration of active enzyme is equal to the total protein concentration in the enzyme preparation and an enzyme mass of 32 kDa.
§No activity detected under the conditions tested (up to 80 μM substrate; up to 6 h incubation).
||Low Vmax precluded accurate estimation of kinetic parameters.
¶Km above the range of the substrate concentrations used in this analysis precluded accurate estimation of kinetic parameters.