Abstract：
:Much interest has centered on two surface loops in the motor domain to explain the differences in enzymatic and mechanical properties of myosin isoforms. We showed that two invariant lysines at the C-terminal end of loop 2, which is part of the actin-binding interface, are required to obtain actin activation [Joel et al. (2001) J. Biol. Chem. 276, 2998-3003]. Here we investigate the effects of increasing positive charge in the variable portion of loop 2 of smooth muscle heavy meromyosin (smHMM). Increasing the net positive charge by +4 increased the affinity for actin in the presence and absence of ATP. The K(m) for actin-activated ATPase activity decreased 15-fold, but V(max) was unchanged, showing that "weak binding" of myosin for actin can be significantly strengthened without increasing the rate-limiting step for V(max). The mutant HMM had slower rates of in vitro motility and ADP release compared to WT HMM. ADP release and motility, which were both salt-dependent, correlated linearly with each other. Loop 2 thus plays a major role in setting the affinity for actin but also affects ADP release and motility. Because the actin- and nucleotide-binding regions communicate, mutations to one region can impact multiple facets of myosin's mechanical and enzymatic properties.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Joel PB,Sweeney HL,Trybus KMdoi
10.1021/bi034415jsubject
Has Abstractpub_date
2003-08-05 00:00:00pages
9160-6issue
30eissn
0006-2960issn
1520-4995journal_volume
42pub_type
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