TABLE 1.
Summary of kinetic and motility data for HMM constructs
| HMM constructs | Scallop structural homologsa | NH4+-ATPaseb | Basal MgATPasec | Vmaxd | KATPased | Motilitye | NVmaxNmotf |
|---|---|---|---|---|---|---|---|
| s−1 head−1 | s−1 | s−1 | μm | μm s−1 | |||
| WT | 7.6 ± 0.1 | 0.032, 0.032 | 2.7 ± 0.7 | 38 ± 8 | 0.25 ± 0.02 | 4,5 | |
| M129Q (RLC) | Met-116 | 7.1 ± 0.1 | 0.021, 0.018 | 0.35 ± 0.05 | <10 | 0 | 2,3 |
| G130C (RLC) | Gly-117 | 7.1 ± 0.2 | 0.024, 0.025 | 0.38 ± 0.04 | <10 | 0 | 2,3 |
| M129Q/G130C (RLC) | Met-116/Gly-117 | 7.1 ± 0.1 | 0.017, 0.010 | 1.2 ± 0.1 | 10 ± 3 | 0 | 2,2 |
| M129Q/G130C/R20 M (RLC-ELC) | Met-116/Gly-117/Trp-21 | 7.1 ± 0.1 | 0.037, 0.021 | 1.4 ± 0.2 | 45 ± 15 | 0.11 ± 0.00 | 2,2 |
| M129Q/G130C/K25A (RLC-ELC) | Met-116/Gly-117/Ala-27 | 7.1 ± 0.1 | 0.012, 0.012 | 1.8 ± 0.5 | 28 ± 4 | 0.13 ± 0.04 | 2,4 |
| R20M (ELC) | g | 7.1 ± 0.1 | 0.019, 0.012 | 2.8 ± 0.1 | 50 ± 0 | 0.22 ± 0.02 | 2,2 |
| K25A (ELC) | Ala-27 | 7.1 ± 0.1 | 0.030, 0.017 | 2.7 ± 0.3 | 46 ± 2 | 0.21 ± 0.03 | 2,2 |
| D131A (RLC) | Asp-118 | 7.5 ± 0.3 | 0.035, 0.033 | 2.5 ± 0.3 | 37 ± 3 | 0.23 ± 0.00 | 2,2 |
| Q826A (HC) | Gln-812 | 7.5 ± 0.3 | 0.017, 0.011 | 2.5 ± 0.3 | 30 ± 6 | 0.25 ± 0.02 | 3,2 |
| Q826A/D131A (HC-RLC) | Gln-816/Asp-118 | 7.5 ± 0.3 | 0.032, 0.024 | 0.75 ± 0.2 | 13 ± 5 | 0.07 ± 0.02 | 2,2 |
| E13A (ELC) | Asp-13 | 7.5 ± 0.3 | 0.010, 0.026 | 1.2 ± 0.1 | 59 ± 22 | 0.11 ± 0.01 | 2,2 |
| Q816A (HC) | Gln-802 | 7.5 ± 0.3 | 0.024, 0.021 | 2.3 ± 0.2 | 71 ± 15 | 0.16 ± 0.04 | 2,3 |
| Q816A/E13A (HC-ELC) | Gln-802/Asp-13 | 7.5 ± 0.3 | 0.029, 0.029 | 0.97 ± 0.09 | 25 ± 8 | 0.05 ± 0.01 | 2,2 |
| R827M (HC) | Arg-813 | 7.5 ± 0.3 | 0.032, 0.034 | 2.5 ± 0.3 | 52 ± 4 | 0.15 ± 0.03 | 2,2 |
| N828A (HC) | Asn-814 | 7.5 ± 0.3 | 0.031, 0.039 | 2.1 ± 0.03 | 19 ± 4 | 0.22 ± 0.02 | 2,2 |
| L819A/M822A/Q826A (HC) | Arg-805/Leu-808/Gln-812 | ND | 0.011, 0.017 | 2.7 ± 0.1 | 48 ± 8 | 0.24 ± 0.03 | 2,2 |
a Data are based upon PDB code 1QVI (scallop). The HC of the smooth muscle myosin structure (PDB code 1BR1) is not solved beyond sm821, so the structural homologs of 822, 826, 827, and 828 are extrapolated and are therefore estimates.
b Steady-state NH4+-ATPase rates for the uP-state in the absence of actin are shown.
c Basal MgATPase rates for the P-state (absence of actin) are shown, but otherwise are measured under identical conditions for samples used to determine the Vmax (see Footnote d). Two independently determined values are shown. Values for the uP-state ranged between 0.010 and 0.030 for all constructs studied (data not shown).
d To determine Vmax and KATPase, P-HMM was assayed as described (22, 27) by actin-activated single-turnover of 2′-(3′)-O-(N-methylanthraniloyl)-ATP. See under “Experimental Procedures” for definitions of variables, fitting equations, number of determinations, and meaning of the indicated errors.
e Data are for the P-state. No motility was detected for any of the mutants in the uP-state (data not shown).
f NVmax and Nmot refer to the number of times that independent measurements of Vmax and motility were performed on independent samples prepared from different batches of cells.
g RLC homologs are based upon sequence alignment. ELC homologs for Glu-13 and Lys-25 are based upon structural alignment using smooth PDB code 1BR4. Arg-20 in smooth is in a loop that is two amino acids shorter than in scallop (21WDG23). Therefore, there is no unequivocal structural homolog.