慈菇蛋白酶抑制剂A和B的活性中心探讨

慈菇蛋白酶抑制A和B（APIA和APIB）是一种双头多功能抑制剂。它们的一级结构和cDNA序列已经被阐明。为了找到它们的活性中心，利用定点诱变的方法将APIB中根据与其他抑制剂家族的序列比较所推断的可能的活性中心残基；Lys^44,Arg^76和Arg87分别用Pro替代，所得到的突变基因分别在酵母分泌体系中得到了表达，与天然的APIB相比，K^44P-APIB对脂蛋白酶的抑制活力没有改变；而R^76P-APIB和R^87P-APIB对胰蛋白酶的抑制活力都分别下降了一半，由原料的抑制两分子变成了一分子，表明Arg^76和Arg^87分别是APIB的两个活性中心残基，而Lys^44则不是，为了证实以上结论，进一步制备了另外3种突变体（K^44P-R^76P-APIB,K^44P-R^87P-APIB,R^76P-R^87P-APIB)。在每个突变体中，3个可能的活性位点中只保留1个，有关的抑制活力测定表明，K^44P-R^76P-APIB(只保留Arg^87)和K^44P-R^87P-APIB(只保留Arg^76)分别只抑制一分子胰蛋白酶，而R^76P-R^87P-APIB(只保留Lys^44)对胰蛋白酶基本不抑制，从而肯定了以上结论，经过测定，两个突变体K^44P-R^87P-APIB对胰蛋白酶的抑制常数Ki分别是0.39nmol/L和0.47nmol/L。突变体R^87L-APIB(APIA中87位是Leu)丧失了接近一半的胰蛋白酶抑制活力，但同时对胰凝乳蛋白酶的抑制活性由原来的基本不抑制变成和APIA相同的可以抑制一分子，证明了Leu^87是APIA的抑制胰凝乳蛋白酶的活性中心位点。

The Assignment of the Reactive Sites of the Double-headed Arrowhead Proteinase Inhibitor A and B

The arrowhead proteinase inhibitor A and B (APIA and APIB) are double headed and multifunctional. Both their primary structure and cDNA sequence have been elucidated. To locate the possible reactive site residues Lys 44 , Arg 76 and Arg 87 of APIB predicted according to the sequence comparison with other proteinase inhibitors, the above residues were substituted with Pro by site directed mutagenesis respectively, and the mutated genes were expressed in the yeast secretion system. The mutant K 44 P APIB displayed the same inhibitory activity as APIB does, while the mutants R 76 P APIB and R 87 P APIB could only inhibit one molecule, instead of two molecules of trypsin, indicating that Arg 76 and Arg 87 but not Lys 44 are the two reactive sites of APIB. In order to further confirm this result, more mutants of APIB (K 44 P R 76 P APIB, K 44 P R 87 P APIB, R 76 P R 87 P APIB) were designed, in each mutant only one of the three possible reactive sites remained unchanged. Both the mutants K 44 P R 76 P APIB and K 44 P R 87 P APIB could only inhibit one molecule of trypsin, while R 76 P R 87 P APIB could no longer inhibit trypsin intensively, thus the residues Arg 76 and Arg 87 are definitely the reactive sites of APIB. Their K i were measured to be 0.39 nmol/L and 0.47 nmol/L, respectively. The mutant R 87 L APIB lost about half activity towards trypsin but could inhibit one molecule of chymotrypsin as the wide type APIA did, indicating that Leu 87 was the reactive site towards trypsin but could inhibit one molecule of of APIA for inhibiting chymotrypsin.