Petithory J R, Masiarz F R, Kirsch J F, Santi D V, Malcolm B A
Department of Molecular and Cell Biology, University of California, Berkeley.
Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11510-4. doi: 10.1073/pnas.88.24.11510.
The preferred amino acid residues at the P'1 and P'2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P'1 or P'2 positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termini, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative kcat/Km values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P'1 mixture were glycine, alanine, and serine. The enzyme has little specificity at P'2; only arginine and proline peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases.
通过一种快速筛选方法确定了甲型肝炎病毒3C蛋白酶（HAV-3C）肽底物P'1和P'2位置上的优选氨基酸残基。用两种N端乙酰化肽的单独混合物处理该酶，这两种混合物除了P'1或P'2位置的氨基酸外序列相同，在这些位置引入了一组15或16种氨基酸。肽混合物的酶催化水解产生游离氨基末端，这使得能够通过埃德曼降解进行直接序列分析。在适当的测序循环中每种氨基酸产物的相对产率给出了每种底物混合物组分水解的量。这允许同时评估混合物中每种组分的相对kcat/Km值。HAV-3C蛋白酶在P'1混合物中优选的肽底物是甘氨酸、丙氨酸和丝氨酸。该酶在P'2处特异性很小；只有精氨酸和脯氨酸肽被排除作为底物。该方法可快速确定肽底物的优选残基，并且应该适用于其他内切蛋白酶。
