Davis J H, Agard D A
Graduate Group in Biophysics, Howard Hughes Medical Institute, University of California, San Francisco 94143-0448, USA.
Biochemistry. 1998 May 26;37(21):7696-707. doi: 10.1021/bi972963p.
To better understand the structural basis for the observed patterns in substrate specificity, the backbone dynamics of alpha-lytic protease have been investigated using 15N relaxation measurements. The enzyme was inhibited with the peptide boronic acid N-tert-butyloxycarbonyl-Ala-Pro-boroVal [Kettner, C. A., et al. (1988) Biochemistry 27, 7682], which mimics interactions occurring in the tetrahedral transition state or nearby intermediates, and the dynamics of the unbound and inhibited enzyme were compared. Arrayed 2-D NMR spectra were acquired to measure T1, T2, and steady-state ¿1H¿-15N NOE of >95% of the backbone amides in both protein samples. The overall rotational correlation time tauc was found to be 8.1 ns. Values of the spectral density function J(omega) at omega = 0, omegaN, and approximately omegaH were derived from the relaxation results using reduced spectral density mapping [Ishima, R., & Nagayama, K. (1995) Biochemistry 34, 3162]. The resultant spectral densities were interpreted to indicate regions of fast motion (nanosecond to picosecond) and of intermediate chemical exchange (millisecond to microsecond). The protein has 13 regions with increased motion on the fast time scale; these generally fall on exterior turns and loops and most correlate with regions of higher crystallographic B-factors. Several stretches of backbone undergo intermediate chemical exchange, indicating motion or other processes that cause temporal chemical shift changes. A comparison of spectral densities for both the free and inhibited enzymes revealed that inhibitor binding preferentially stabilizes regions undergoing chemical exchange (which predominate around the active site) and only minimally affect regions of rapid motion. Slow motions, suggestive of backbone plasticity, are observed in most of the binding pocket residues. This may point to a mechanism for the observed broad specificity of the enzyme. The significance of the observed dynamics for substrate binding and specificity is discussed.
为了更好地理解所观察到的底物特异性模式的结构基础，已使用15N弛豫测量研究了α-溶菌酶蛋白酶的主链动力学。该酶用肽硼酸N-叔丁氧羰基-Ala-Pro-硼缬氨酸抑制[Kettner, C. A.,等人(1988)《生物化学》27,7682]，其模拟在四面体过渡态或附近中间体中发生的相互作用，并比较了未结合和受抑制酶的动力学。采集了二维核磁共振谱阵列，以测量两种蛋白质样品中>95%的主链酰胺的T1、T2和稳态1H-15N NOE。发现整体旋转相关时间tauc为8.1 ns。使用简化谱密度映射[Ishima, R., & Nagayama, K. (1995)《生物化学》34, 3162]从弛豫结果中得出ω = 0、ωN和近似ωH时的谱密度函数J(ω)值。对所得谱密度的解释表明了快速运动区域（纳秒到皮秒）和中间化学交换区域（毫秒到微秒）。该蛋白质在快速时间尺度上有13个运动增加的区域；这些区域通常位于外部转角和环上，并且大多与较高晶体学B因子的区域相关。几段主链经历中间化学交换，表明存在导致时间化学位移变化的运动或其他过程。对游离酶和受抑制酶的谱密度比较表明，抑制剂结合优先稳定经历化学交换的区域（主要在活性位点周围），并且对快速运动区域的影响最小。在大多数结合口袋残基中观察到暗示主链可塑性的缓慢运动。这可能指出了该酶观察到的广泛特异性的一种机制。讨论了所观察到的动力学对底物结合和特异性的意义。
