TABLE 3.
Sequence information, catalytic and structural properties, and MD simulations of pseudolysin, vibriolysin MCP-02, and vibriolysin E495
| Enzyme | Pseudolysin | Vibriolysin MCP-02 | Vibriolysin E495 |
|---|---|---|---|
| Sequence | |||
| Precursor/mature sequence lengtha | 498/301 (301) | 727/315 (302) | 730/310 (302) |
| Pairwise identity MCP-02 | 59.5% | ||
| Pairwise identity E495 | 58.9% | 90.7% | |
| Leu number (ratio) | 14 (4.65%) | 20 (6.62%) | 18 (5.96%) |
| Ile number (ratio) | 9 (2.99%) | 7 (2.32%) | 7 (2.32%) |
| Val number (ratio) | 19 (6.31%) | 16 (5.30%) | 16 (5.30%) |
| Asn number (ratio) | 20 (6.64%) | 34 (11.3%) | 32 (10.6%) |
| Arg number (ratio) | 15 (4.98%) | 5 (1.66%) | 4 (1.32%) |
| Ser number (ratio) | 25 (8.31%) | 28 (9.27%) | 33 (10.9%) |
| Thr number (ratio) | 18 (5.98%) | 16 (5.30%) | 21 (6.95%) |
| Tyr number (ratio) | 22 (7.31%) | 18 (5.96%) | 17 (5.63%) |
| Trp number (ratio) | 4 (1.33%) | 6 (1.99%) | 6 (1.99%) |
| His number (ratio) | 7 (2.33%) | 5 (1.66%) | 5 (1.66%) |
| Net charges (Arg+Lys-Asp-Glu)b | –5 | –13 | –12 |
| Catalytic and structural properties | |||
| Optimal pH (casein, 60 °C)c | 7.5 | 8.0 | 7.5 |
| Topt (casein, optimal pH)d | 62 °C | 57 °C | 57 °C |
| kcat/Km (FAGLA, 25 °C) (s–1 mm–1)e | 0.64 ± 0.01 | 1.32 ± 0.01 | 2.55 ± 0.05 |
| Half-life t½ (55 °C) (min)f | 530 | 90 | 82 |
| Half-life t½ (60 °C) (min)f | 160 | 23 | 20 |
| Inactivation rate kinact (55 °C) (s–1)f | 2.2 × 10–5 | 1.3 × 10–4 | 1.4 × 10–4 |
| Inactivation rate kinact (60 °C) (s–1)f | 7.3 × 10–5 | 5.1 × 10–4 | 5.8 × 10–4 |
| Apparent Tm (fluorescence)g | 75 °C | 64 °C | 63 °C |
| Apparent Tm (CD)g | 72 °C | 65 °C | 65 °C |
| ΔKSV (40–15 °C, acrylamide) (m–1)h | 2.6 ± 0.1 | 5.3 ± 0.1 | 5.7 ± 0.1 |
| MD simulations (280 K/310 K)i | |||
| Mean r.m.s.f. (backbone) (Å) | 0.65/0.84 | 0.67/0.88 | 0.78/0.93 |
| Total HBs | 750/925 | 778/1023 | 887/1117 |
| Unstable HBs (persistency < 5%) | 367/500 | 429/621 | 484/680 |
| Average HBs in each frame | 219 ± 6/206 ± 7 | 221 ± 7/210 ± 7 | 219 ± 7/209 ± 7 |
| Average HB persistency | 29.2%/22.3% | 28.4%/20.5% | 24.7%/18.7% |
| Total salt bridges | 23/26 | 14/15 | 16/18 |
| Average salt bridges in each frame | 13.4 ± 0.8/14.4 ± 1.1 | 8.1 ± 1.0/8.1 ± 1.0 | 5.8 ± 0.9/8.0 ± 1.0 |
| Average salt bridge persistency | 58%/55% | 58%/54% | 36%/44% |
The numbers in parentheses are the residues used in the calculation of sequence identity and amino acid composition
Because each enzyme contains one zinc ion and one calcium ion, the total number of charges for each enzyme will be –1 for pseudolysin, –9 for vibriolysin MCP-02, and –8 for vibriolysin E495
See supplemental Fig. S4
The experiments were carried out at the optimal pH for each enzyme with 1% (w/w) casein as the substrate. See Fig. 4A
The pH was 8.0. See Fig. 3
The experiments were carried out at the optimal pH for each enzyme with 1% (w/w) casein as the substrate
The experiments were carried out at pH 8.0. See Fig. 4 (B and C)
The increase in the Stern-Volmer quenching constant KSV from 15 °C to 40 °C. The slopes of lines are presented in Fig. 5
The r.m.s.f., HB, and salt bridge data were calculated from the last 15-ns data of 30-ns simulations and are presented in the form of 280 K/310 K