title: Synthesis of a novel fluorescent non-nucleotide ATP analogue and its interaction with myosin ATPase.
abstract: A novel non-nucleotide fluorescent ATP analogue, N-methylanthraniloylamideethyl triphosphate (MANTTP), was designed and synthesized for kinetic studies with ATPases. The interaction of MANTTP with myosin ATPase was characterized. MANTTP was used as a substrate of myosin ATPase, and acceleration of actin-dependent hydrolysis was observed. The fluorescence property of MANTTP was not greatly affected by its binding to the ATPase site of myosin. In contrast, during MANTTP hydrolysis, significant fluorescence resonance energy transfer (FRET) was observed between MANTTP and intrinsic tryptophan residues in the myosin motor domain. Binding of MANTTP and formation of a ternary complex with a myosin-N-methylanthraniloylamideethyl diphosphate (MANTDP)-Pi analogue, which may mimic ATPase transient states, were monitored by FRET. The kinetic parameters of MANTTP binding to myosin and MANTDP release from the ATPase site were determined using a stopped-flow apparatus and compared with those of other ATP analogues. This novel fluorescent ATP analogue was shown to be applicable for kinetic analysis of ATPases.
authors.affiliation: Division of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tanaka
authors.forename: Taro
authors.initials: T
authors.lastname: Kimura
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Maruta
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000201:enzymology; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000199:Actins; C569810:N-methylanthraniloylamideethyl triphosphate; D000255:Adenosine Triphosphate; D009218:Myosins