Kinetic mechanism of myosin IXB and the contributions of two class IX-specific regions.
Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.
Nalavadi
Vijayalaxmi
V
Institute for General Zoology and Genetics, Westfalian Wilhelms-University, 48149 Münster, Germany.
Nyitrai
Miklós
M
Bertolini
Cristina
C
Adamek
Nancy
N
Geeves
Michael A
MA
Bähler
Martin
M
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D000199:Actins; D018076:DNA, Complementary; D020409:Molecular Motor Proteins; D011994:Recombinant Proteins; C113469:myosin IXB; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins