Dinakarpandian D, Shenoy B, Pusztai-Carey M, Malcolm B A, Carey P R
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Biochemistry. 1997 Apr 22;36(16):4943-8. doi: 10.1021/bi963148x.
Although the HAV 3C proteinase is a cysteine protease, it displays an active site configuration which resembles mammalian serine proteases and is structurally distinct from the papain superfamily of thiol proteases. Given the interesting serine/cysteine protease hybrid nature of HAV 3C, we have probed its active site properties via the Raman spectra of the acyl enzyme, 5-methylthiophene acryloyl HAV 3C, using the C24S variant of the enzyme to obtain stoichiometric acylation. The Raman difference spectral data show that the major population of the acyl groups in the active site experiences electron polarization intermediate between that in the papain superfamily and that in a nonpolarizing site. This is evidenced by the values of the acyl group ethylenic stretching frequency which occur near 1602 cm(-1) in a nonpolarizing environment, at 1588 cm(-1) when bound to HAV 3C (C24S), and at 1579 cm(-1) in acyl papains. The value of the electronic absorption maximum for the HAV 3C (C24S) acyl enzyme and the deacylation rate constant fit the correlation developed for the papain superfamily, suggesting that for HAV 3C too, polarizing forces in the active site can contribute to rate acceleration via transition state stabilization. The major population in the active site is s-cis about the acyl group's C1-C2 bond, but there is a second population that is s-trans, and this secondary population is not polarized. The two populations are evidenced by the presence of two sets of marker bands for s-cis and s-trans in the Raman spectra, which occur principally in the C=C stretching region near 1600 cm(-1), in the C-C stretching region near 1100 cm(-1), and near 560 cm(-1). The positions of the acyl carbonyl features in the Raman spectra point to hydrogen-bonding strengths of 20-25 kJ mol(-1) between the C=O and H-bonding donors in the active site. The 5-methylthiophene acryloyl HAV 3C (C24S) is a relatively unreactive acyl enzyme, deacylating with a pKa of 7.1 and a rate constant of 0.000 31 s(-1) at pH 9. Unlike most other cysteine or serine protease acyl enzymes characterized by Raman spectroscopy, no changes in the Raman spectrum could be detected with changes in pH.
虽然甲型肝炎病毒3C蛋白酶是一种半胱氨酸蛋白酶，但其活性位点结构类似于哺乳动物丝氨酸蛋白酶，在结构上与硫醇蛋白酶的木瓜蛋白酶超家族不同。鉴于甲型肝炎病毒3C有趣的丝氨酸/半胱氨酸蛋白酶杂合性质，我们通过酰基酶5-甲基噻吩丙烯酰甲型肝炎病毒3C的拉曼光谱，利用该酶的C24S变体获得化学计量酰化，来探究其活性位点性质。拉曼差光谱数据表明，活性位点中主要的酰基群体经历的电子极化介于木瓜蛋白酶超家族和非极化位点之间。这由酰基乙烯基伸缩频率的值证明，该值在非极化环境中接近1602 cm⁻¹，与甲型肝炎病毒3C（C24S）结合时为1588 cm⁻¹，在酰基木瓜蛋白酶中为1579 cm⁻¹。甲型肝炎病毒3C（C24S）酰基酶的电子吸收最大值和脱酰化速率常数符合为木瓜蛋白酶超家族建立的相关性，这表明对于甲型肝炎病毒3C而言，活性位点中的极化力也可通过过渡态稳定作用促进速率加速。活性位点中的主要群体围绕酰基的C1 - C2键呈s - 顺式，但存在第二个呈s - 反式的群体，且该次要群体未极化。这两个群体由拉曼光谱中s - 顺式和s - 反式的两组标记带证明，它们主要出现在1600 cm⁻¹附近的C = C伸缩区域、1100 cm⁻¹附近的C - C伸缩区域以及560 cm⁻¹附近。拉曼光谱中酰基羰基特征的位置表明活性位点中C = O与氢键供体之间的氢键强度为20 - 25 kJ mol⁻¹。5 - 甲基噻吩丙烯酰甲型肝炎病毒3C（C24S）是一种相对不活泼的酰基酶，在pH 9时脱酰化的pKa为7.1，速率常数为0.000 31 s⁻¹。与大多数其他通过拉曼光谱表征的半胱氨酸或丝氨酸蛋白酶酰基酶不同，随着pH变化，未检测到拉曼光谱有变化。
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