TABLE 5.
Kinetic, size-exclusion HPLC, and MALDI-TOF experiments probing the interaction of E1p and E3p with the E2p component and its variants
| Substitutions in E2 | Overall activity | Interaction between E1p and E2pa | Interaction between E3 and E2pa | Reductive acetylation of the E2b |
|
|---|---|---|---|---|---|
| Reductively acetylated lipoyl domainc | Unacetylated lipoyl domainc | ||||
| % | |||||
| None | 100 | + | + | + | − |
| R129A | 34 | + | − | + | + |
| R129K | 77 | + | + | + | + |
| R129E | 3.0 | − | − | − | + |
| R130A | 100 | NA | NA | NA | NA |
| R130E | 57 | NA | NA | NA | NA |
| R150A | 8.0 | − | + | NA | NA |
| R150K | 5.0 | − | + | + | + |
| R150E | 2.4 | − | − | − | + |
| R153K | 83 | + | + | + | + |
| R153E | 60 | + | + | + | + |
| K161A | 65 | NA | NA | + | + |
| K161E | 84 | NA | NA | NA | NA |
| K191R | 79 | NA | NA | NA | NA |
| K191E | 50 | NA | NA | NA | NA |
| R202K | 86 | + | + | + | + |
| R202E | 10 | + | + | NA | NA |
a + indicates E2 variants converted quantitatively to E1p-E2p or E3-E2p subcomplexes according to size-exclusion HPLC experiment; − indicates E2 variants that did not form the subcomplexes with E1p or E3 according to size-exclusion HPLC experiment. NA means data were not available.
b The reductive acetylation of the E2p lipoyl domain was monitored by MALDI-TOF MS as presented under “Experimental Procedures.”
c The molecular mass of the acetylated lipoyl domain = 10,154 Da and that of the unacetylated lipoyl domain = 10,110 Da.