We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-P i (inorganic phosphate) and actomyosin V-ADP-P i and the rate of the hydrolysis step (myosin V-ATP 3 myosin V-ADP-P i ) were all ϳ10-fold slower in the S217A mutant than in wild type (WT) myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from ϳ0.85 in WT myosin V to ϳ0.25 in S217A and produces a motor in which the average run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, myosin V can be converted into a low duty ratio motor that is processive only at low substrate concentrations.During the past 2 decades a considerable number of different myosins have been discovered (1). Myosin V is the best characterized among the so-called unconventional myosins (i.e. those not belonging to class II), and it serves as an important model molecule for studying actomyosin interactions and single molecule processive motility (2). Myosin V is a highly processive motor whose role is to transport cargo along actin filaments or bundles inside the cell (3-5). The kinetic mechanism of myosin V is significantly different from that of conventional myosins such as muscle myosin II, as it remains bound to actin (filamentous actin) through a number of ATPase cycles (6 -8). Myosin V has a high duty ratio: a single-headed myosin V-S1 (myosin V, subfragment 1) is in the strongly bound AM-ADP state 80 -90% of the time during ATP hydrolysis. An additional mechanism for promoting highly processive runs is the preferential release of ADP from the trail head because of mechanochemical gating, which causes a drastic reduction of the rate constant of ADP release from the lead head (9 -11). Although there are significant differences between the ATPase mechanisms of the different myosins, the structure of the nucleotide binding pocket (composed of the switch 1 and 2 regions and the P-loop) is highly conserved. The position of the Ser 217 (Ser 236 in Dictyostelium myosin II) residue of the switch 1 loop (the first serine in the NDNSSRFG sequence) is shown in Fig. 1. It had been shown previously by mutagenesis in Dictyostelium (12) and in smooth muscle myosin II (13) that the substitution of serine 236 to alanine retains at least partial enzymatic and motile function in these mutant myosins. Therefore, the OH group is not an essential part of the catalytic mechanism, but the rate of steady-state ATP hydrolysis is reduced several fold. However, neither of these studies includes a detailed kinetic analysis to det...

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3-(7-diethylaminocoumarin-3-carbonylamino)-3-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. Myosin V is an unconventional myosin that transports organelles such as vesicles in neurons and melanosomes in melanocytes along actin tracks (1). To do this, myosin V has evolved the ability to move processively on actin for several m, requiring many ATP hydrolysis cycles (2). The processivity is at least partially explained by a kinetic mechanism in which the slow rate of ADP dissociation is the rate-limiting step of ATP hydrolysis (3,4). This is in contrast to the myosin II mechanism, in which ADP dissociation is much faster and dissociation of actin from myosin occurs with each cycle of ATP hydrolysis. As a result, myosin V has a much higher duty cycle, and even single headed myosin V is in the strongly bound actomyosin-ADP state 80 -90% of the time during steady-state hydrolysis (3). It has also been suggested that additional mechanisms promoting processivity may involve gating of product release (5, 6). Thus, biochemical cycles of the lead and trail head keep out of synchrony, so one head of the molecule is always strongly attached (actomyosin or actomyosin-ADP). Biochemical (7), mechanical (5, 6, 8), and structural studies (9, 10) suggest that intramolecular strain accelerates ADP dissociation from the trail head and/or inhibits ADP dissociation from the lead head. The long lever arm of myosin V, which contains six calmodulin or light chain molecules, produces a 20 -25-nm power stroke that can be observed as two substeps of 15-20 and 5 nm (11,12).Biochemical evidence showing the interaction between the two heads is from kinetic data obtained by Rosenfeld and Sweeney (7), who observed biphasic dissociation of mdADP 5 from actomyosin V-HMM. In these single-mixing stoppedflow experiments, the rates of mdADP dissociation were measured by mixing myosin-mdADP complexes with actin in the presence of either a large excess of ATP or ADP that can act as a chase reagent, binding to nucleotide-free sites as they form. An alternative approach to measure the post-power stroke dissociation of ADP resulting from a single turnover of ATP hydrolysis is to use double-mixing stopped-flow measurements. In these experiments, the myosin is first mixed with ATP (or fluorescent analogues), incubated for a few seconds to allow the ATP to bind to the myosin and be hydrolyzed, and then mixed with actin. A power stroke is associated with P i dissociation from act...

We have used a new fluorescent ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine-5'-triphosphate (deac-aminoATP), to study the ATP hydrolysis mechanism of the single headed myosinV-S1. Our study demonstrates that deac-aminoATP is an excellent substrate for these studies. Although the deac-amino nucleotides have a low quantum yield in free solution, there is a very large increase in fluorescence emission ( approximately 20-fold) upon binding to the myosinV active site. The fluorescence emission intensity is independent of the hydrolysis state of the nucleotide bound to myosinV-S1. The very good signal-to-noise ratio that is obtained with deac-amino nucleotides makes them excellent substrates for studying expressed proteins that can only be isolated in small quantities. The combination of the fast rate of binding and the favorable signal-to-noise ratio also allows deac-nucleotides to be used in chase experiments to determine the kinetics of ADP and Pi dissociation from actomyosin-ADP-Pi. Although phosphate dissociation from actomyosinV-ADP-Pi does not itself produce a fluorescence signal, it produces a lag in the signal for deac-aminoADP dissociation. The lag provides direct evidence that the principal pathway of product dissociation from actomyosinV-ADP-Pi is an ordered mechanism in which phosphate precedes ADP. Although the mechanism of hydrolysis of deac-aminoATP by (acto)myosinV-S1 is qualitatively similar to the ATP hydrolysis mechanism, there are significant differences in some of the rate constants. Deac-aminoATP binds 3-fold faster to myosinV-S1, and the rate of deac-aminoADP dissociation from actomyosinV-S1 is 20-fold slower. Deac-aminoATP supports motility by myosinV-HMM on actin at a rate consistent with the slower rate of deac-aminoADP dissociation.

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These preliminary findings permitted the identification of several "maturational antigens" for cynomolgus monkey spermatozoa. Further characterization of these antigens that are modified during epididymal transit is warranted to determine their significance in the acquisition of progressive motility and fertilizing ability by epididymal spermatozoa.

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