Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis

被引:25
作者
Ajtai, K
Peyser, YM
Park, S
Burghardt, TP [1 ]
Muhlrad, A
机构
[1] Mayo Clin & Mayo Fdn, Dept Biochem & Mol Biol, Rochester, MN 55905 USA
[2] Hebrew Univ Jerusalem, Hadassah Sch Dent Med, Dept Oral Biol, IL-91120 Jerusalem, Israel
关键词
D O I
10.1021/bi990149r
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (SI) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim, Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of SI [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+-and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.
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收藏
页码:6428 / 6440
页数:13
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