Table 2.

Transient kinetic parameters of the actomyosin-7a ATPase cycle with corresponding values for rodent and Drosophila class 7 myosins

Parameter	Signal or calculation	Hs Myosin-7a	Mm Myosin-7a [47]	Dm Myosin-7a [48]	Dm Myosin-7a [42]	Dm Myosin-7b [50]	Mm Myosin-7b [46]
K 1 k +2 (μM−1s_1)	mantATP	1.15 ± 0.01	2.5 ± 0.04 (dmATP)	n.d.	3.4 ± 0.2	3.4 ± 0.1 (dmATP)	1.8 ± 0.1
	Tryptophan	0.85 ± 0.02	2.2	n.d.	2.6 ± 0.2	4.7	4.1 ± 0.9
K 1 k +2 (μM−1s−1)	Light scattering	0.44 ± 0.01	n.d.	1.6 (pyrene signal)	0.35 ± 0.04	n.d.	1.4 ± 0.1
	mantATP	0.68 ± 0.01	0.53 ± 0.04 (dmATP)	1.0 ± 0.1 (dmATP)	0.47 ± 0.02	2.2, 0.46
1/K 1 (μM)	Light scattering	681.19 ± 5.63	295 ± 42	93	n.d.	n.d.	646 ± 100
k +2 (s−1)	Light scattering	186 ± 5.70	350 ± 19	148 ± 27	>200	>400, >50 (pyrene-actin)	729 ± 46
k 3 + k -3 (s−1)	Tryptophan	23.1 ± 0.74	11.5 ± 0.83	10.6 ± 0.4	12.6 ± 0.9	160 ± 30	318 ± 23
k +D (μM−1s−1)	mantADP	0.98 ± 0.05	1.2 ± 0.06 (dmADP)	n.d.	3.7 ± 0.1	2.3 ± 0.1 (dmADP)	3.1 ± 0.1
k −D (s−1)	mantADP	0.27 ± 0.01a		n.d.	1.8 ± 0.3	9.0 ± 0.2 (dmADP)	4.5 ± 1.6
	mantADP	0.20 ± 0.01b	2.1 ± 0.07 (dmADP)	n.d.	2.3 ± 0.1	10.1 ± 0.7 (dmADP)
	Tryptophan	0.30 ± 0.002	n.d.	n.d.	n.d.	n.d.
K D (μM)	Tryptophan	0.52 ± 0.09	3.5 (dmADP)e	n.d.	n.d.	n.d.	0.2 ± 0.05
	k −D/k +D	0.27 ± 0.03	1.75 (dmADP)	n.d.	0.62 ± 0.03	3.8	1.3 ± 0.5
k +AD (μM−1s−1)	mantADP	1.78 ± 0.04	1.5 ± 0.09 (dmADP)	3.9 ± 0.1 (dmADP)	4.7 ± 0.2	2.7 ± 0.4 (dmADP)	3.4 ± 0.2
k −AD (s−1)	mantADP	0.59 ± 0.03a	n.d.	n.d.	2.3 ± 0.5	16 ± 1 (dmADP)	7.0 ± 2.2
	Light scattering	0.48 ± 0.01b	1.28 ± 0.09 (ADP)	7.7 ± 0.8 (pyrene data)	1.86 ± 0.03	9.8 ± 3.5 (pyrene-actin)	n.d.
	mantADP	0.32 ± 0.01b	1.7 ± 0.2 (dmADP)	7.8 ± 2.3 (dmADP)	2.1 ± 0.5	9.6 ± 1.3 (dmADP)	6.9 ± 0.1
K i (Mg2+free) (mM)	mantADP	0.29 ± 0.03b	n.d.	n.d.	n.d.	n.d.	n.d.
K AD (μM)	k −AD/k +AD	0.30 ± 0.22 (mantADP)	1 (dmADP)	n.d.	0.48 ± 0.127	3.6-5.9 (dmADP)	2
Coupling*	K AD/K D	~1.2	~0.3–0.6	n.d.	~0.8	~0.7–1.6	~10
Coupling**	k −AD/k −D	~1.1–1.8	~0.6–0.8	n.d.	~1	~1.6	~1.5
k +A (μM−1s−1)	Pyrene-actin	3.17 ± 0.06d	4.7 ± 0.25 (light scattering)	n.d.	0.88 ± 0.03	1.2 ± 0.1	12.1 ± 2.7
k −A (s−1)	Pyrene-actin	0.002 ± 4·10−5 d	n.d.	n.d.	<0.0043	0.042	0.054 ± 0.0002
K A (nM)	k −A/k +A	0.63 ± 0.02	n.d.	n.d.	4.8	35	4.5 ± 1.0
k +DA (μM−1 s−1)	Pyrene-actin	1.34 ± 0.03d	2.6 ± 0.25 (light scattering)	n.d.	0.32 ± 0.02	1.0 ± 0.1	16.3 ± 6.0
k −DA (s−1)	Pyrene-actin	0.003 ± 1·10−4 d	n.d.	n.d.	<0.03	0.034	0.043 ± 0.0001
K DA (nM)	k −DA/k +DA	2 ± 0.13	n.d.	n.d.	94	34	2.6 ± 1.0
Coupling***	K DA/K A	~3.17	n.d.	n.d.	~19.5	~0.97	~0.57
Duty ratio		~0.6c	0.6	0.88	~0.9	~0.8	~0.8

Numbering of the kinetic rates and constants correspond to Scheme 1. Experimental conditions were as follows if not stated otherwise: 25 mM MOPS (pH 7.0), 100 mM KCl, and 5 mM MgCl₂

* Thermodynamic coupling relating the affinity of ADP for actomyosin with the affinity of ADP for myosin

** ADP release rate enhancement by F-actin

*** Thermodynamic coupling relating the affinity of F-actin for myosin·ADP with the affinity of F-actin for myosin

^aOrdinate of the k _obs versus [mantADP] plot

^bATP-chase experiment

^c5 mM MgCl₂, as calculated from duty ratio = ((1/k _−AD) + (1/k ₊₂))/(1/k _cat)

^dMOPS buffer supplemented with 10 mM DTT

^eSingle turnover data