DiSanto M E, Li Q H, Logan D A
Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104.
J Bacteriol. 1992 Jan;174(2):447-55. doi: 10.1128/jb.174.2.447-455.1992.
A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.
从二态真菌总状毛霉的菌丝体中纯化出一种受发育调控的羧肽酶。该酶被命名为羧肽酶3（CP3），已被纯化至同质且纯化倍数超过900倍，并对其进行了表征。在等电聚焦聚丙烯酰胺凝胶电泳（PAGE）中，羧肽酶迁移为单一电泳条带，其等电点为pH 4.4。通过凝胶过滤估计天然酶的表观分子量为52 kDa。在非还原条件下进行的十二烷基硫酸钠（SDS）-PAGE显示存在一条51 kDa的单一多肽。用2-巯基乙醇处理CP3后的SDS-PAGE显示存在两条分别为31 kDa和18 kDa的多肽，表明该酶具有由二硫键连接亚基组成的二聚体结构（α1β1）。通过使用[1,3-3H]二异丙基氟磷酸作为活性位点标记试剂，确定催化位点位于羧肽酶的小亚基上。以N-苄氧羰基-L-苯丙氨酰-L-亮氨酸（N-CBZ-Phe-Leu）为底物时，Km、kcat和Vmax值分别为1.7×10⁻⁴ M、490 s⁻¹以及每毫克蛋白质每分钟释放588 μmol亮氨酸。CP3被确定为一种丝氨酸蛋白酶，因为其催化活性被丝氨酸蛋白酶抑制剂二异丙基氟磷酸、苯甲基磺酰氟和3,4-二氯异香豆素（DCI）所阻断。该酶被汞化合物对氯汞苯甲酸强烈抑制。羧肽酶能轻易水解具有脂肪族或芳香族侧链的肽，而大多数在倒数第二位含有甘氨酸的肽不能作为该酶的底物。尽管在总状毛霉酵母细胞中未检测到CP3活性，但抗血清显示在该真菌的酵母形态中存在该酶。已确定了羧肽酶的部分氨基酸序列。
