Li X D, Rhodes T E, Ikebe R, Kambara T, White H D, Ikebe M
Department of Physiology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655-0127, USA.
J Biol Chem. 1998 Oct 16;273(42):27404-11. doi: 10.1074/jbc.273.42.27404.
The role of the highly conserved residues in the gamma-phosphate binding site of myosin upon myosin motor function was studied. Each of five residues (Ser181, Lys185, Asn235, Ser236, and Arg238) in smooth muscle myosin was mutated. K185Q has neither a steady state ATPase nor an initial Pi burst. Although ATP and actin bind to K185Q, it is not dissociated from actin by ATP. These results indicate that the hydrolysis of bound ATP by K185Q is inhibited. S236T has nearly normal basal Mg2+-ATPase activity, initial Pi burst, ATP-induced enhancement of intrinsic tryptophan fluorescence, and ATP-induced dissociation from actin. However, the actin activation of the Mg2+-ATPase activity and actin translocation of S236T were blocked. In contrast S236A has nearly normal enzymatic properties and actin-translocating activity. These results indicate that 1) the hydroxyl group of Ser236 is not critical as an intermediary of proton transfer during the ATP hydrolysis step, and 2) the bulk of the extra methyl group of the threonine residue in S236T blocks the acceleration of product release from the active site by actin. Arg238, which interacts with Glu459 at the Switch II region, was mutated to Lys and Ile, respectively. R238K has essentially normal enzymatic activity and motility. In contrast, R238I does not hydrolyze ATP or support motility, although it still binds ATP. These results indicate that the charge interaction between Glu459 and Arg238 is critical for ATP hydrolysis by myosin. Other mutants, S181A, S181T, and N235I, showed nearly normal enzymatic and motile activity.
研究了肌球蛋白γ-磷酸结合位点中高度保守的残基在肌球蛋白运动功能中的作用。对平滑肌肌球蛋白中的五个残基（Ser181、Lys185、Asn235、Ser236和Arg238）分别进行了突变。K185Q既没有稳态ATP酶活性，也没有初始Pi爆发。尽管ATP和肌动蛋白能与K185Q结合，但它不会被ATP从肌动蛋白上解离。这些结果表明K185Q对结合的ATP的水解受到抑制。S236T具有近乎正常的基础Mg2+-ATP酶活性、初始Pi爆发、ATP诱导的内在色氨酸荧光增强以及ATP诱导的从肌动蛋白上解离。然而，Mg2+-ATP酶活性的肌动蛋白激活以及S236T的肌动蛋白转位被阻断。相比之下，S236A具有近乎正常的酶学性质和肌动蛋白转位活性。这些结果表明：1）Ser236的羟基在ATP水解步骤中作为质子转移的中间体并不关键；2）S236T中苏氨酸残基额外甲基的大部分阻碍了肌动蛋白加速产物从活性位点释放。与开关II区域的Glu459相互作用的Arg238分别突变为Lys和Ile。R238K具有基本正常的酶活性和运动性。相比之下，R238I虽然仍能结合ATP，但不水解ATP也不支持运动。这些结果表明Glu459和Arg238之间的电荷相互作用对肌球蛋白的ATP水解至关重要。其他突变体S181A、S181T和N235I表现出近乎正常的酶活性和运动活性。
