Brown M T, McGrath W J, Toledo D L, Mangel W F
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
FEBS Lett. 1996 Jun 17;388(2-3):233-7. doi: 10.1016/0014-5793(96)00569-8.
The type of proteinase and the nature of the active site of the human adenovirus proteinase are unknown. For these reasons we produced an inhibitor profile of the enzyme. Enzyme activity in disrupted virions was inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. Of the inhibitors that worked, the most useful potentially in illuminating the nature of the active site was bovine pancreatic trypsin inhibitor (BPTI), and for this reason we extensively characterized the interaction with BPTI. In disrupted virions, the enzyme is irreversibly inhibited by BPTI with a Ki of 35 nM and a ki of 6.2 x 10(-4) s(-1). One reason enzyme activity is inhibited is that BPTI, a basic protein, precipitates the viral DNA, a cofactor of enzyme activity. In vitro with purified components, BPTI acts as a competitive inhibitor (Ki 2 microM) of the recombinant proteinase complexed with its 11-amino-acid cofactor pVIc. The recombinant endoproteinase is beat labile whereas its 11-amino-acid cofactor is heat stable. We estimate there are about 50 molecules of proteinase per virus particle.
人类腺病毒蛋白酶的蛋白酶类型和活性位点的性质尚不清楚。基于这些原因，我们对该酶进行了抑制剂谱分析。几种丝氨酸特异性和半胱氨酸特异性蛋白酶抑制剂可抑制破碎病毒颗粒中的酶活性。在起作用的抑制剂中，最有可能有助于阐明活性位点性质的是牛胰蛋白酶抑制剂（BPTI），因此我们对其与BPTI的相互作用进行了广泛表征。在破碎病毒颗粒中，该酶被BPTI不可逆抑制，Ki为35 nM，ki为6.2×10⁻⁴ s⁻¹。酶活性受到抑制的一个原因是，作为碱性蛋白质的BPTI会沉淀病毒DNA，而病毒DNA是酶活性的一种辅助因子。在体外使用纯化成分时，BPTI作为与11个氨基酸的辅助因子pVIc复合的重组蛋白酶的竞争性抑制剂（Ki为2 μM）。重组内蛋白酶对热不稳定，而其11个氨基酸的辅助因子对热稳定。我们估计每个病毒颗粒约有50个蛋白酶分子。
