Table 1.

Steady-state and transient kinetic parameters of WT and D179Y myosin VI-MD^ins2

Kinetic parameters	MVI-MDins2 WT	MVI-MDins2 (D179Y)
Steady-state parameters
VMAX,* s−1	6.6 ± 0.3	1.9 ± 0.4
KATPase,* µM	12.4 ± 0.2	NA
V0 (−actin),*,† s−1	0.11 ± 0.1	1.2 ± 0.6
Rate constants of the actomyosin VI ATPase cycle (Scheme 1)
k+4A′: Pi release with actin,‡ s−1	121 ± 4.2	133 ± 8.3
k+4A: Pi release (−actin),‡ s−1	0.3 ± 0.1	58.1 ± 10.3
k+4B′: Weak-to-strong transition on actin (k+4B′)§	28.3 ± 0.58	38.6 ± 3.1
k+5’: ADP release from actomyosin VI,¶ s−1	7.0 ± 0.08	2.03 ± 0.07
k-5’: ADP binding to actomyosin VI,¶ µM−1⋅s−1	0.57 ± 0.29	0.51 ± 0.10
k+5: ADP release (−actin),¶ s−1	8.5 ± 0.18	2.6 ± 0.18
k-5: ADP binding (−actin),¶ µM−1⋅s−1	1.2 ± 0.34	0.49 ± 0.17
ATP-induced dissociation from pyrene-actin
k+2′,§ s−1	568 ± 24.9	79.1 ± 5.1
K1′k+2′,§ mM−1⋅s−1	13.9 ± 1.2	14.2 ± 3.0

NA, not assessed.

^*Measured in the NADH coupled assay.

^†Steady-state turnover in the absence of actin.

^‡Measured using phosphate-binding protein (PiBiP).

^§Measured with pyrene actin.

^¶Measured with mantADP.